Archaeal Viruses Research Articles

Structural studies of Acidianus tailed spindle virus reveal a structural paradigm used in the assembly of spindle-shaped viruses

The spindle-shaped virion morphology is common among archaeal viruses, where it is a defining characteristic of many viral families. However, structural heterogeneity intrinsic to spindle-shaped viruses has seriously hindered efforts to elucidate the molecular architecture of these lemon-shaped capsids. We have utilized a combination of cryo-electron microscopy and X-ray crystallography to study Acidianus tailed spindle virus (ATSV). These studies reveal the architectural principles that underlie assembly of a spindle-shaped virus. Cryo-electron tomography shows a smooth transition from the spindle-shaped capsid into the tubular-shaped tail and allows low-resolution structural modeling of individual virions. Remarkably, higher-dose 2D micrographs reveal a helical surface lattice in the spindle-shaped capsid. Consistent with this, crystallographic studies of the major capsid protein reveal a decorated four-helix bundle that packs within the crystal to form a four-start helical assembly with structural similarity to the tube-shaped tail structure of ATSV and other tailed, spindle-shaped viruses. Combined, this suggests that the spindle-shaped morphology of the ATSV capsid is formed by a multistart helical assembly with a smoothly varying radius and allows construction of a pseudoatomic model for the lemon-shaped capsid that extends into a tubular tail. The potential advantages that this novel architecture conveys to the life cycle of spindle-shaped viruses, including a role in DNA ejection, are discussed.

Taxonomy of prokaryotic viruses: 2017 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

Structure and assembly mechanism of virus-associated pyramids.

Viruses have developed intricate molecular machines to infect, replicate within and escape from their host cells. Perhaps one of the most intriguing of these mechanisms is the pyramidal egress structure that has evolved in archaeal viruses, such as SIRV2 or STIV1. The structure and mechanism of these virus-associated pyramids (VAPs) has been studied by cryo-electron tomography and complementary biochemical techniques, revealing that VAPs are formed by multiple copies of a virus-encoded 10-kDa protein (PVAP) that integrate into the cell membrane and assemble into hollow, sevenfold symmetric pyramids. In this process, growing VAPs puncture the protective surface layer and ultimately open to release newly replicated viral particles into the surrounding medium. PVAP has the striking capability to spontaneously integrate and self-assemble into VAPs in biological membranes of the archaea, bacteria and eukaryotes. This renders the VAP a universal membrane remodelling system. In this review, we provide an overview of the VAP structure and assembly mechanism and discuss the possible use of VAPs in nano-biotechnology.

Phylogenomics of Cas4 family nucleases

BackgroundThe Cas4 family endonuclease is a component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. Unlike most of the other Cas proteins, Cas4 is often encoded outside CRISPR-cas loci (solo-Cas4) and is also found in mobile genetic elements (MGE-Cas4).ResultsAs part of our ongoing investigation of CRISPR-Cas evolution, we explored the phylogenomics of the Cas4 family. About 90% of the archaeal genomes encode Cas4 compared to only about 20% of the bacterial genomes. Many archaea encode both the CRISPR-associated form (CAS-Cas4) and solo-Cas4, whereas in bacteria, this combination is extremely rare. The solo-cas4 genes are over-represented in environmental bacteria and archaea with small genomes that typically lack CRISPR-Cas, suggesting that Cas4 could perform uncharacterized defense or repair functions in these microbes. Phylogenomic analysis indicates that both the CRISPR-associated cas4 genes are often transferred horizontally but almost exclusively, as part of the adaptation module. The evolutionary integrity of the adaptation module sharply contrasts the rampant shuffling of CRISPR-cas modules whereby a given variant of the adaptation module can combine with virtually any effector module. The solo-cas4 genes evolve primarily via vertical inheritance and are subject only to occasional horizontal transfer. The selection pressure on cas4 genes does not substantially differ between CAS-Cas4 and solo-cas4, and is close to the genomic median. Thus, cas4 genes, similarly to cas1 and cas2, evolve similarly to ‘regular’ microbial genes involved in various cellular functions, showing no evidence of direct involvement in virus-host arms races. A notable feature of the Cas4 family evolution is the frequent recruitment of cas4 genes by various mobile genetic elements (MGE), particularly, archaeal viruses. The functions of Cas4 in these elements are unknown and potentially might involve anti-defense roles.ConclusionsUnlike most of the other Cas proteins, Cas4 family members are as often encoded by stand-alone genes as they are incorporated in CRISPR-Cas systems. In addition, cas4 genes were repeatedly recruited by MGE, perhaps, for anti-defense functions. Experimental characterization of the solo and MGE-encoded Cas4 nucleases is expected to reveal currently uncharacterized defense and anti-defense systems and their interactions with CRISPR-Cas systems.

Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging

Archaeal viruses have evolved to infect hosts often thriving in extreme conditions such as high temperatures. However, there is a paucity of information on archaeal virion structures, genome packaging, and determinants of temperature resistance. The rod-shaped virus APBV1 (Aeropyrum pernix bacilliform virus 1) is among the most thermostable viruses known; it infects a hyperthermophile Aeropyrum pernix, which grows optimally at 90 °C. Here we report the structure of APBV1, determined by cryo-electron microscopy at near-atomic resolution. Tight packing of the major virion glycoprotein (VP1) is ensured by extended hydrophobic interfaces, and likely contributes to the extreme thermostability of the helical capsid. The double-stranded DNA is tightly packed in the capsid as a left-handed superhelix and held in place by the interactions with positively charged residues of VP1. The assembly is closed by specific capping structures at either end, which we propose to play a role in DNA packing and delivery.

The enigmatic archaeal virosphere.

One of the most prominent features of archaea is the extraordinary diversity of their DNA viruses. Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic viruses and represent unique virus families. The distinct nature of archaeal viruses also extends to the gene composition and architectures of their genomes and the properties of the proteins that they encode. Environmental research has revealed prominent roles of archaeal viruses in influencing microbial communities in ocean ecosystems, and recent metagenomic studies have uncovered new groups of archaeal viruses that infect extremophiles and mesophiles in diverse habitats. In this Review, we summarize recent advances in our understanding of the genomic and morphological diversity of archaeal viruses and the molecular biology of their life cycles and virus-host interactions, including interactions with archaeal CRISPR-Cas systems. We also examine the potential origins and evolution of archaeal viruses and discuss their place in the global virosphere.

ICTV Virus Taxonomy Profile: Pleolipoviridae.

Members of the family Pleolipoviridae (termed pleolipoviruses) are pseudo-spherical and pleomorphic archaeal viruses. The enveloped virion is a simple membrane vesicle, which encloses different types of DNA genomes of approximately 7–16 kbp (or kilonucleotides). Typically, virions contain a single type of transmembrane (spike) protein at the envelope and a single type of membrane protein, which is embedded in the envelope and located in the internal side of the membrane. All viruses infect extremely halophilic archaea in the class Halobacteria (phylum Euryarchaeota). Pleolipoviruses have a narrow host range and a persistent, non-lytic life cycle. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Pleolipoviridae which is available at www.ictv.global/report/pleolipoviridae.

Bacteriophage Taxonomy: An Evolving Discipline.

While taxonomy is an often-unappreciated branch of science it serves very important roles. Bacteriophage taxonomy has evolved from a mainly morphology-based discipline, characterized by the work of David Bradley and Hans-Wolfgang Ackermann, to the holistic approach that is taken today. The Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) takes a comprehensive approach to classifying prokaryote viruses measuring overall DNA and protein identity and phylogeny before making decisions about the taxonomic position of a new virus. The huge number of complete genomes being deposited with NCBI and other public databases has resulted in a reassessment of the taxonomy of many viruses, and the future will see the introduction of new viral families and higher orders.

Methanosarcina Spherical Virus, a Novel Archaeal Lytic Virus Targeting Methanosarcina Strains.

A novel archaeal lytic virus targeting species of the genus Methanosarcina was isolated using Methanosarcina mazei strain Gö1 as the host. Due to its spherical morphology, the virus was designated Methanosarcina spherical virus (MetSV). Molecular analysis demonstrated that MetSV contains double-stranded linear DNA with a genome size of 10,567 bp containing 22 open reading frames (ORFs), all oriented in the same direction. Functions were predicted for some of these ORFs, i.e., such as DNA polymerase, ATPase, and DNA-binding protein as well as envelope (structural) protein. MetSV-derived spacers in CRISPR loci were detected in several published Methanosarcina draft genomes using bioinformatic tools, revealing a potential protospacer-adjacent motif (PAM) motif (TTA/T). Transcription and expression of several predicted viral ORFs were validated by reverse transcription-PCR (RT-PCR), PAGE analysis, and liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Analysis of core lipids by atmospheric pressure chemical ionization (APCI) mass spectrometry showed that MetSV and Methanosarcina mazei both contain archaeol and glycerol dialkyl glycerol tetraether without a cyclopentane moiety (GDGT-0). The MetSV host range is limited to Methanosarcina strains growing as single cells (M. mazei, Methanosarcina barkeri and Methanosarcina soligelidi). In contrast, strains growing as sarcina-like aggregates were apparently protected from infection. Heterogeneity related to morphology phases in M. mazei cultures allowed acquisition of resistance to MetSV after challenge by growing cultures as sarcina-like aggregates. CRISPR/Cas-mediated resistance was excluded since neither of the two CRISPR arrays showed MetSV-derived spacer acquisition. Based on these findings, we propose that changing the morphology from single cells to sarcina-like aggregates upon rearrangement of the envelope structure prevents infection and subsequent lysis by MetSV.IMPORTANCE Methanoarchaea are among the most abundant organisms on the planet since they are present in high numbers in major anaerobic environments. They convert various carbon sources, e.g., acetate, methylamines, or methanol, to methane and carbon dioxide; thus, they have a significant impact on the emission of major greenhouse gases. Today, very little is known about viruses specifically infecting methanoarchaea that most probably impact the abundance of methanoarchaea in microbial consortia. Here, we characterize the first identified Methanosarcina-infecting virus (MetSV) and show a mechanism for acquiring resistance against MetSV. Based on our results, we propose that growth as sarcina-like aggregates prevents infection and subsequent lysis. These findings allow new insights into the virus-host relationship in methanogenic community structures, their dynamics, and their phase heterogeneity. Moreover, the availability of a specific virus provides new possibilities to deepen our knowledge of the defense mechanisms of potential hosts and offers tools for genetic manipulation.

SMV1, an extremely stable thermophilic virus platform for nanoparticle trafficking in the mammalian GI tract.

Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract. A major challenge of using nano-vectors to target gut microbiome is their survival during passage through the extremely acidic and proteolytic environment of the mammalian GI tract. Here, we investigated the thermo-acidophilic archaeal virus SMV1 as a candidate therapeutic nano-vector for the distal mammalian GI tract microbiome. We investigated the anatomical distribution, vector stability and immunogenicity of this virus following oral ingestion in mice and compared these traits to the more classically used Inovirus vector M13KE. We found that SMV1 particles were highly stable under both simulated GI tract conditions (invitro) and in mice (invivo). Moreover, SMV1 could not be detected in tissues outside the GI tract and it elicited a nearly undetectable inflammatory response. Finally, we used human intestinal organoids (HIOs) to show that labelled SMV1 did not invade or otherwise perturb the human GI tract epithelium. Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract. This is the first study evaluating an archaeal virus as a potential therapeutic nanoparticle delivery system and it opens new possibilities for future development of novel nanoplatforms.

Isolation and Characterization of Metallosphaera Turreted Icosahedral Virus, a Founding Member of a New Family of Archaeal Viruses.

Our understanding of archaeal virus diversity and structure is just beginning to emerge. Here we describe a new archaeal virus, tentatively named Metallosphaera turreted icosahedral virus (MTIV), that was isolated from an acidic hot spring in Yellowstone National Park, USA. Two strains of the virus were identified and were found to replicate in an archaeal host species closely related to Metallosphaera yellowstonensis Each strain encodes a 9.8- to 9.9-kb linear double-stranded DNA (dsDNA) genome with large inverted terminal repeats. Each genome encodes 21 open reading frames (ORFs). The ORFs display high homology between the strains, but they are quite distinct from other known viral genes. The 70-nm-diameter virion is built on a T=28 icosahedral lattice. Both single particle cryo-electron microscopy and cryotomography reconstructions reveal an unusual structure that has 42 turret-like projections: 12 pentameric turrets positioned on the icosahedral 5-fold axes and 30 turrets with apparent hexameric symmetry positioned on the icosahedral 2-fold axes. Both the virion structural properties and the genome content support MTIV as the founding member of a new family of archaeal viruses.IMPORTANCE Many archaeal viruses are quite different from viruses infecting bacteria and eukaryotes. Initial characterization of MTIV reveals a virus distinct from other known bacterial, eukaryotic, and archaeal viruses; this finding suggests that viruses infecting Archaea are still an understudied group. As the first known virus infecting a Metallosphaera sp., MTIV provides a new system for exploring archaeal virology by examining host-virus interactions and the unique features of MTIV structure-function relationships. These studies will likely expand our understanding of virus ecology and evolution.

Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice

Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties.

Marine archaea and archaeal viruses under global change.

Global change is altering oceanic temperature, salinity, pH, and oxygen concentration, directly and indirectly influencing marine microbial food web structure and function. As microbes represent >90% of the ocean’s biomass and are major drivers of biogeochemical cycles, understanding their responses to such changes is fundamental for predicting the consequences of global change on ecosystem functioning. Recent findings indicate that marine archaea and archaeal viruses are active and relevant components of marine microbial assemblages, far more abundant and diverse than was previously thought. Further research is urgently needed to better understand the impacts of global change on virus–archaea dynamics and how archaea and their viruses can interactively influence the ocean’s feedbacks on global change.

DNA-Interacting Characteristics of the Archaeal Rudiviral Protein SIRV2_Gp1.

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod-shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function. The SIRV2_Gp1 protein is highly expressed during early stages of infection and it is the only protein that is encoded twice on the viral genome. It harbours a helix-turn-helix motif and was therefore hypothesized to bind DNA. The DNA-binding behavior of SIRV2_Gp1 was characterized with electrophoretic mobility shift assays and atomic force microscopy. We provide evidence that the protein interacts with DNA and that it forms large aggregates, thereby causing extreme condensation of the DNA. Furthermore, the N-terminal domain of the protein mediates toxicity to the viral host Sulfolobus. Our findings may lead to biotechnological applications, such as the development of a toxic peptide for the containment of pathogenic bacteria, and add to our understanding of the Rudiviral infection cycle.

VICTOR: genome-based phylogeny and classification of prokaryotic viruses

MotivationBacterial and archaeal viruses are crucial for global biogeochemical cycles and might well be game-changing therapeutic agents in the fight against multi-resistant pathogens. Nevertheless, it is still unclear how to best use genome sequence data for a fast, universal and accurate taxonomic classification of such viruses.ResultsWe here present a novel in silico framework for phylogeny and classification of prokaryotic viruses, in line with the principles of phylogenetic systematics, and using a large reference dataset of officially classified viruses. The resulting trees revealed a high agreement with the classification. Except for low resolution at the family level, the majority of taxa was well supported as monophyletic. Clusters obtained with distance thresholds chosen for maximizing taxonomic agreement appeared phylogenetically reasonable, too. Analysis of an expanded dataset, containing >4000 genomes from public databases, revealed a large number of novel species, genera, subfamilies and families.Availability and implementationThe selected methods are available as the easy-to-use web service ‘VICTOR’ at https://victor.dsmz.de.Supplementary information Supplementary data are available at Bioinformatics online.

Sulfolobus Spindle-Shaped Virus 1 Growth Kinetics

Geothermal and hypersaline environments are rich in viral particles, among which spindle-shaped morphologies predominate. Currently, viruses with spindle- or lemon-shaped virions are unique to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae, encompasses spindle-shaped viruses with very short tails, which infect Sulfolobus solfataricus and close relatives. Sulfolobus spindle-shaped virus 1 (SSV1) is the best-known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. However, our knowledge of fuselloviral life cycles and the relationships between these viruses and their hosts is still limited. As a result, to establish one-step growth curves to study virus replication kinetics and to investigate the mechanism of host binding, the adsorption kinetics of wild-type SSV1 to its Sulfolobus host needed to be determined.

Putative archaeal viruses from the mesopelagic ocean.

Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce “MArVD”, for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

Formation of a Viral Replication Focus in Sulfolobus Cells Infected by the Rudivirus Sulfolobus islandicus Rod-Shaped Virus 2.

Viral factories are compartmentalized centers for viral replication and assembly in infected eukaryotic cells. Here, we report the formation of a replication focus by prototypical archaeal Sulfolobus islandicus rod-shaped virus 2 (SIRV2) in the model archaeon Sulfolobus This rod-shaped virus belongs to the viral family Rudiviridae, carrying linear double-stranded DNA (dsDNA) genomes, which are very common in geothermal environments. We demonstrate that SIRV2 DNA synthesis is confined to a focus near the periphery of infected cells. Moreover, viral and cellular replication proteins are recruited to, and concentrated in, the viral replication focus. Furthermore, we show that of the four host DNA polymerases (DNA polymerase I [Dpo1] to Dpo4), only Dpo1 participates in viral DNA synthesis. This constitutes the first report of the formation of a viral replication focus in archaeal cells, suggesting that organization of viral replication in foci is a widespread strategy employed by viruses of the three domains of life.IMPORTANCE The organization of viral replication in foci or viral factories has been mostly described for different eukaryotic viruses and for several bacteriophages. This work constitutes the first report of the formation of a viral replication center by a virus infecting members of the Archaea domain.

A Novel Type of Polyhedral Viruses Infecting Hyperthermophilic Archaea.

Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated.IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, "Portogloboviridae."

VConTACT: an iVirus tool to classify double-stranded DNA viruses that infect Archaea and Bacteria.

Taxonomic classification of archaeal and bacterial viruses is challenging, yet also fundamental for developing a predictive understanding of microbial ecosystems. Recent identification of hundreds of thousands of new viral genomes and genome fragments, whose hosts remain unknown, requires a paradigm shift away from traditional classification approaches and towards the use of genomes for taxonomy. Here we revisited the use of genomes and their protein content as a means for developing a viral taxonomy for bacterial and archaeal viruses. A network-based analytic was evaluated and benchmarked against authority-accepted taxonomic assignments and found to be largely concordant. Exceptions were manually examined and found to represent areas of viral genome ‘sequence space’ that are under-sampled or prone to excessive genetic exchange. While both cases are poorly resolved by genome-based taxonomic approaches, the former will improve as viral sequence space is better sampled and the latter are uncommon. Finally, given the largely robust taxonomic capabilities of this approach, we sought to enable researchers to easily and systematically classify new viruses. Thus, we established a tool, vConTACT, as an app at iVirus, where it operates as a fast, highly scalable, user-friendly app within the free and powerful CyVerse cyberinfrastructure.