bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.907410; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 New virus isolates from Italian hydrothermal environments underscore the biogeographic 2 pattern in archaeal virus communities 3 4 5 6 Diana P. Baquero1,2, Patrizia Contursi3, Monica Piochi4, Simonetta Bartolucci3, Ying Liu1, 7 Virginija Cvirkaite-Krupovic1, David Prangishvili1,* and Mart Krupovic1,* 8 9 10 1 Archaeal Virology Unit, Department of Microbiology, Institut Pasteur, 75015 Paris, France 11 2 Sorbonne Universités, UPMC Univ, 75005 Paris, France 12 3 University of Naples Federico II, Department of Biology, Naples, Italy 13 4 Istituto Nazionale di Geofisica e Vulcanologia, Naples, Osservatorio Vesuviano, Italy 14 15 * Correspondence to: [email protected] and [email protected] 16 Institut Pasteur, Department of Microbiology, 17 75015 Paris, France 18 Tel: 33 (0)1 40 61 37 22 19 20 21 Running title 22 Biogeographic pattern in archaeal virus communities 23 24 25 Competing Interests 26 The authors declare that they have no competing interests. 27 28 29 30 31 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.907410; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 ABSTRACT 33 Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, 34 showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we 35 investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, 36 Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, 37 droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed 38 to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and 39 characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus 40 Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family 41 Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the 42 order Sulfolobales, namely, Saccharolobus, Acidianus and Metallosphaera. Notably, 43 Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. 44 Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear 45 biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting 46 geographical structuring of virus communities in extreme geothermal environments. Furthermore, 47 we propose a revised classification of the Rudiviridae family, with establishment of five new 48 genera. Collectively, our results further show that high-temperature continental hydrothermal 49 systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses. 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.907410; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 INTRODUCTION 51 One of the most remarkable features of hyperthermophilic archaea is the diversity and uniqueness 52 of their viruses. Most of these viruses infect members of the phylum Crenarchaeota and are 53 evolutionarily unrelated to viruses infecting bacteria, eukaryotes or even archaea thriving at 54 moderate temperate [1-4]. Thus far, unique to hyperthermophilic archaea are rod-shaped viruses 55 of the families Rudiviridae and Clavaviridae; filamentous enveloped viruses of the families 56 Lipothrixviridae and Tristromaviridae; as well as spherical (Globuloviridae), ellipsoid 57 (Ovaliviridae), droplet-shaped (Guttaviridae), coil-shaped (Spiraviridae) and bottle-shaped 58 (Ampullaviridae) viruses [1,5]. Hyperthermophilic archaea are also infected by two types of 59 spindle-shaped viruses, belonging to the families Fuselloviridae and Bicaudaviridae [6,7]. 60 Whereas bicaudaviruses appear to be restricted to hyperthermophiles, viruses distantly related to 61 fuselloviruses are also known to infect hyperhalophilic archaea [8], marine hyperthermophilic 62 archaea [9,10] and marine ammonia-oxidizing archaea from the phylum Thaumarchaeota [11]. 63 Finally, hyperthermophilic archaea are also infected by three groups of viruses with icosahedral 64 virions, Turriviridae [12], Portogloboviridae [13] and two closely related, unclassified viruses 65 infecting Metallosphaera species [14]. Portoglobovirus SPV1 is structurally and genomically 66 unrelated to other known viruses [13,15], whereas viruses structurally similar to turriviruses are 67 widespread in all three domains of life [1,16,17]. Structural studies on filamentous and spindle- 68 shaped crenarchaeal viruses have illuminated the molecular details of virion organization and 69 further underscored the lack of relationship to viruses of bacteria and eukaryotes [18-24]. 70 71 The uniqueness of hyperthermophilic archaeal viruses extends to their genomes, with ∼75% of the 72 genes lacking detectable homologues in sequence databases [25]. All characterized archaeal 73 viruses have DNA genomes, which can be single-stranded (ss) or double-stranded (ds), linear or 74 circular. Comparative genomic and bipartite network analyses have shown that viruses of 75 hyperthermophilic archaea share only few genes with the rest of the virosphere [26]. Furthermore, 76 most of the genes in viruses from different families are family-specific, albeit a handful of genes 77 encoding transcription regulators, glycosyltransferases and anti-CRISPR proteins display broader 78 distribution [27]. These observations, in combination with the structural studies, led to the 79 suggestion that crenarchaeal viruses have originated on multiple independent occasions and 80 constitute a unique part of the virosphere [1]. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.01.16.907410; this version posted January 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 81 82 Although the infection cycles of crenarchaeal viruses has been studied for just a handful of 83 representative viruses, the available data has already provided valuable insight into the virus-host 84 interaction strategies in archaea. For instance, members of the Fuselloviridae, Guttaviridae, 85 Turriviridae and Bicaudaviridae are temperate and their genomes can be site-specifically 86 integrated into the host chromosome by means of a virus-encoded integrase [28-33]. In the case of 87 SSV1, the most extensively characterized member of the Fuselloviridae family, the virus 88 integrates its genome into the host chromosome at an arginyl-tRNA gene upon cell infection. After 89 exposure to UV light, the excision and replication of the SSV1 viral genomes is induced, leading 90 to virion release [34,35]. Two different egress strategies have been elucidated. The enveloped 91 virions of fusellovirus SSV1 are assembled at the host cell membrane and are released from the 92 cell by a budding mechanism similar to that of some eukaryotic enveloped viruses [36]. By 93 contrast, lytic crenarchaeal viruses belonging to three unrelated families, Rudiviridae, Turriviridae 94 and Ovaliviridae, employ a unique release mechanism based on the formation of pyramidal 95 protrusions on the host cell surface, leading to perforation of the cell envelope and release of 96 intracellularly assembled mature virions [5,37,38]. 97 98 Single-cell sequencing combined with environmental metagenomics of hydrothermal microbial 99 community from Yellowstone National Park [39] led to the estimation that >60% of cells contain 100 at least one virus type and a majority of these cells contain two or more virus types [40]. However, 101 despite their diversity, distinctiveness and abundance, the number of isolated species of viruses 102 infecting hyperthermophilic archaea remains low compared to the known eukaryotic or bacterial 103 viruses [2]. Indeed, it has been estimated that only about 0.01-0.1% of viruses present in 104 geothermal acidic environments have been isolated [41]. Similarly, using a combination of viral 105 assemblage sequencing and network analysis, it has been estimated that out of 110 identified virus 106 groups, less than 10% represent known archaeal viruses, suggesting that the vast majority of virus 107 clusters represent unknown viruses, likely infecting archaeal hosts [42]. Furthermore, the evolution 108 and structuring of virus communities in terrestrial hydrothermal settings remains poorly 109 understood. Here, to improve understanding on these issues, we explored the diversity of archaeal 110 viruses at the active solfataric field of the Campi Flegrei volcano [43,44] in Pozzuoli, Italy, namely 111 the Pisciarelli hydrothermal area. We report