Hiding in plain sight: The discovery of complete genomes of 11 hypothetical spindle-shaped viruses that putatively infect mesophilic ammonia-oxidizing archaea.

The genome of a putative Nitrosopumilaceae virus with a hypothetical spindle-shaped particle morphology was identified in the Yangshan Harbour metavirome from the East China Sea through protein similarity comparison and structure analysis. This discovery was accompanied by a set of 10 geographically dispersed close relatives found in the environmental virus datasets from typical locations of ammonia-oxidizing archaeon distribution. Its host prediction was supported by iPHoP prediction and protein sequence similarity. The structure of the predicted major capsid protein, together with the overall N-glycosylation site, the transmembrane helices prediction, the hydrophilicity profile, and the docking simulation of the major capsid proteins, indicate that these viruses resemble spindle-shaped viruses. It suggests a similarly assembled structure and, consequently, a possibly spindle-shaped morphology of these newly discovered archaeal viruses.

Molecular basis for inhibition of type III-B CRISPR-Cas by an archaeal viral anti-CRISPR protein.

Despite a wide presence of type III clustered regularly interspaced short palindromic repeats, CRISPR-associated (CRISPR-Cas) in archaea and bacteria, very few anti-CRISPR (Acr) proteins inhibiting type III immunity have been identified, and even less is known about their inhibition mechanism. Here, we present the discovery of a type III CRISPR-Cas inhibitor, AcrIIIB2, encoded by Sulfolobus virus S. islandicus rod-shaped virus 3 (SIRV3). AcrIIIB2 inhibits type III-B CRISPR-Cas immune response to protospacers encoded in middle/late-expressed viral genes. Investigation of the interactions between S. islandicus type III-B CRISPR-Cas Cmr-α-related proteins and AcrIIIB2 reveals that the Acr does not bind to Csx1 but rather interacts with the Cmr-α effector complex. Furthermore, in vitro assays demonstrate that AcrIIIB2 can block the dissociation of cleaved target RNA from the Cmr-α complex, thereby inhibiting the Cmr-α turnover, thus preventing host cellular dormancy and further viral genome degradation by the type III-B CRISPR-Cas immunity.

Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome.

Spindle- or lemon-shaped viruses infect archaea in diverse environments. Due to the highly pleomorphic nature of these virions, which can be found with cylindrical tails emanating from the spindle-shaped body, structural studies of these capsids have been challenging. We have determined the atomic structure of the capsid of Sulfolobus monocaudavirus 1, a virus that infects hosts living in nearly boiling acid. A highly hydrophobic protein, likely integrated into the host membrane before the virions assemble, forms 7 strands that slide past each other in both the tails and the spindle body. We observe the discrete steps that occur as the tail tubes expand, and these are due to highly conserved quasiequivalent interactions with neighboring subunits maintained despite significant diameter changes. Our results show how helical assemblies can vary their diameters, becoming nearly spherical to package a larger genome and suggest how all spindle-shaped viruses have evolved from archaeal rod-like viruses.

Diversity, taxonomy, and evolution of archaeal viruses of the class Caudoviricetes.

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

Lytic archaeal viruses infect abundant primary producers in Earth's crust.

The continental subsurface houses a major portion of life's abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we use a combination of metagenomics and virus-targeted direct-geneFISH (virusFISH) to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches display resistances of Ca. Altiarchaea against eight predicted viral clades, which show genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tag and image a putatively viral genome rich in protospacers using fluorescence microscopy. VirusFISH reveals a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

ORF4 of the Temperate Archaeal Virus SNJ1 Governs the Lysis-Lysogeny Switch and Superinfection Immunity.

Recent environmental and metagenomic studies have considerably increased the repertoire of archaeal viruses and suggested that they play important roles in nutrient cycling in the biosphere. However, very little is known about how they regulate their life cycles and interact with their hosts. Here, we report that the life cycle of the temperate haloarchaeal virus SNJ1 is controlled by the product ORF4, a small protein belonging to the antitoxin MazE superfamily. We show that ORF4 controls the lysis-lysogeny switch of SNJ1 and mediates superinfection immunity by repression of genomic DNA replication of the superinfecting viruses. Bioinformatic analysis shows that ORF4 is highly conserved in two SNJ1-like proviruses, suggesting that the mechanisms for lysis-lysogeny switch and superinfection immunity are conserved in this group of viruses. As the lysis-lysogeny switch and superinfection immunity of archaeal viruses have been poorly studied, we suggest that SNJ1 could serve as a model system to study these processes.IMPORTANCE Archaeal viruses are important parts of the virosphere. Understanding how they regulate their life cycles and interact with host cells provide crucial insights into their biological functions and the evolutionary histories of viruses. However, mechanistic studies of the life cycle of archaeal viruses are scarce due to a lack of genetic tools and demanding cultivation conditions. Here, we discover that the temperate haloarchaeal virus SNJ1, which infects Natrinema sp. strain J7, employs a lysis-lysogeny switch and establishes superinfection immunity like bacteriophages. We show that its ORF4 is critical for both processes and acts as a repressor of the replication of SNJ1. These results establish ORF4 as a master regulator of SNJ1 life cycle and provides novel insights on the regulation of life cycles by temperate archaeal viruses and on their interactions with host cells.

Discovery and Characterization of Thermoproteus Spherical Piliferous Virus 1: a Spherical Archaeal Virus Decorated with Unusual Filaments.

We describe the discovery of an archaeal virus, one that infects archaea, tentatively named Thermoproteus spherical piliferous virus 1 (TSPV1), which was purified from a Thermoproteales host isolated from a hot spring in Yellowstone National Park (USA). TSPV1 packages an 18.65-kb linear double-stranded DNA (dsDNA) genome with 31 open reading frames (ORFs), whose predicted gene products show little homology to proteins with known functions. A comparison of virus particle morphologies and gene content demonstrates that TSPV1 is a new member of the Globuloviridae family of archaeal viruses. However, unlike other Globuloviridae members, TSPV1 has numerous highly unusual filaments decorating its surface, which can extend hundreds of nanometers from the virion. To our knowledge, similar filaments have not been observed in any other archaeal virus. The filaments are remarkably stable, remaining intact across a broad range of temperature and pH values, and they are resistant to chemical denaturation and proteolysis. A major component of the filaments is a glycosylated 35-kDa TSPV1 protein (TSPV1 GP24). The filament protein lacks detectable homology to structurally or functionally characterized proteins. We propose, given the low host cell densities of hot spring environments, that the TSPV1 filaments serve to increase the probability of virus attachment and entry into host cells.IMPORTANCE High-temperature environments have proven to be an important source for the discovery of new archaeal viruses with unusual particle morphologies and gene content. Our isolation of Thermoproteus spherical piliferous virus 1 (TSPV1), with numerous filaments extending from the virion surface, expands our understanding of viral diversity and provides new insight into viral replication in high-temperature environments.

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation.

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Structure and mechanisms of viral transcription factors in archaea.

Virus-encoded transcription factors have been pivotal in exploring the molecular mechanisms and regulation of gene expression in bacteria and eukaryotes since the birth of molecular biology, while our understanding of viral transcription in archaea is still in its infancy. Archaeal viruses do not encode their own RNA polymerases (RNAPs) and are consequently entirely dependent on their hosts for gene expression; this is fundamentally different from many bacteriophages and requires alternative regulatory strategies. Archaeal viruses wield a repertoire of proteins to expropriate the host transcription machinery to their own benefit. In this short review we summarise our current understanding of gene-specific and global mechanisms that viruses employ to chiefly downregulate host transcription and enable the efficient and temporal expression of the viral transcriptome. Most of the experimentally characterised archaeo-viral transcription regulators possess either ribbon-helix-helix or Zn-finger motifs that allow them to engage with the DNA in a sequence-specific manner, altering the expression of a specific subset of genes. Recently a novel type of regulator was reported that directly binds to the RNAP and shuts down transcription of both host and viral genes in a global fashion.

Temperature and pH dependence of DNA ejection from archaeal lemon-shaped virus His1.

The archaeal virus His1 isolated from a hypersaline environment infects an extremely halophilic archaeon Haloarcula hispanica. His1 features a lemon-shaped capsid, which is so far found only in archaeal viruses. This unique capsid can withstand high salt concentrations, and can transform into a helical tube, which in turn is resistant to extremely harsh conditions. Hypersaline environments exhibit a wide range of temperatures and pH conditions, which present an extra challenge to their inhabitants. We investigated the influence of pH and temperature on DNA ejection from His1 virus using single-molecule fluorescence experiments. The observed number of ejecting viruses is constant in pH 5 to 9, while the ejection process is suppressed at pH below 5. Similarly, the number of ejections within 15-42 °C shows only a minor increase around 25-37 °C. The maximum velocity of single ejected DNA increases with temperature, in qualitative agreement with the continuum model of dsDNA ejection.

Metaviromics of Namib Desert Salt Pans: A Novel Lineage of Haloarchaeal Salterproviruses and a Rich Source of ssDNA Viruses.

Viral communities of two different salt pans located in the Namib Desert, Hosabes and Eisfeld, were investigated using a combination of multiple displacement amplification of metaviromic DNA and deep sequencing, and provided comprehensive sequence data on both ssDNA and dsDNA viral community structures. Read and contig annotations through online pipelines showed that the salt pans harbored largely unknown viral communities. Through network analysis, we were able to assign a large portion of the unknown reads to a diverse group of ssDNA viruses. Contigs belonging to the subfamily Gokushovirinae were common in both environmental datasets. Analysis of haloarchaeal virus contigs revealed the presence of three contigs distantly related with His1, indicating a possible new lineage of salterproviruses in the Hosabes playa. Based on viral richness and read mapping analyses, the salt pan metaviromes were novel and most closely related to each other while showing a low degree of overlap with other environmental viromes.

A census of α-helical membrane proteins in double-stranded DNA viruses infecting bacteria and archaea.

BACKGROUND: Viruses are the most abundant and genetically diverse biological entities on earth, yet the repertoire of viral proteins remains poorly explored. As the number of sequenced virus genomes grows into the thousands, and the number of viral proteins into the hundreds of thousands, we report a systematic computational analysis of the point of first-contact between viruses and their hosts, namely viral transmembrane (TM) proteins. RESULTS: The complement of α-helical TM proteins in double-stranded DNA viruses infecting bacteria and archaea reveals large-scale trends that differ from those of their hosts. Viruses typically encode a substantially lower fraction of TM proteins than archaea or bacteria, with the notable exception of viruses with virions containing a lipid component such as a lipid envelope, internal lipid core, or inner membrane vesicle. Compared to bacteriophages, archaeal viruses are substantially enriched in membrane proteins. However, this feature is not always stable throughout the evolution of a viral lineage; for example, TM proteins are not part of the common heritage shared between Lipothrixviridae and Rudiviridae. In contrast to bacteria and archaea, viruses almost completely lack proteins with complicated membrane topologies composed of more than 4 TM segments, with the few detected exceptions being obvious cases of relatively recent horizontal transfer from the host. CONCLUSIONS: The dramatic differences between the membrane proteomes of cells and viruses stem from the fact that viruses do not depend on essential membranes for energy transformation, ion homeostasis, nutrient transport and signaling.

Insight into the Assembly of Viruses with Vertical Single ß-barrel Major Capsid Proteins.

Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single ß-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single ß-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly.

Archaeal extrachromosomal genetic elements.

SUMMARY: Research on archaeal extrachromosomal genetic elements (ECEs) has progressed rapidly in the past decade. To date, over 60 archaeal viruses and 60 plasmids have been isolated. These archaeal viruses exhibit an exceptional diversity in morphology, with a wide array of shapes, such as spindles, rods, filaments, spheres, head-tails, bottles, and droplets, and some of these new viruses have been classified into one order, 10 families, and 16 genera. Investigation of model archaeal viruses has yielded important insights into mechanisms underlining various steps in the viral life cycle, including infection, DNA replication and transcription, and virion egression. Many of these mechanisms are unprecedented for any known bacterial or eukaryal viruses. Studies of plasmids isolated from different archaeal hosts have also revealed a striking diversity in gene content and innovation in replication strategies. Highly divergent replication proteins are identified in both viral and plasmid genomes. Genomic studies of archaeal ECEs have revealed a modular sequence structure in which modules of DNA sequence are exchangeable within, as well as among, plasmid families and probably also between viruses and plasmids. In particular, it has been suggested that ECE-host interactions have shaped the coevolution of ECEs and their archaeal hosts. Furthermore, archaeal hosts have developed defense systems, including the innate restriction-modification (R-M) system and the adaptive CRISPR (clustered regularly interspaced short palindromic repeats) system, to restrict invasive plasmids and viruses. Together, these interactions permit a delicate balance between ECEs and their hosts, which is vitally important for maintaining an innovative gene reservoir carried by ECEs. In conclusion, while research on archaeal ECEs has just started to unravel the molecular biology of these genetic entities and their interactions with archaeal hosts, it is expected to accelerate in the next decade.

Comparison of lipid-containing bacterial and archaeal viruses.

Lipid-containing bacteriophages were discovered late and considered to be rare. After further phage isolations and the establishment of the domain Archaea, several new prokaryotic viruses with lipids were observed. Consequently, the presence of lipids in prokaryotic viruses is reasonably common. The wealth of information about how prokaryotic viruses use their lipids comes from a few well-studied model viruses (PM2, PRD1, and ϕ6). These bacteriophages derive their lipid membranes selectively from the host during the virion assembly process which, in the case of PM2 and PRD1, culminates in the formation of protein capsid with an inner membrane, and for ϕ6 an outer envelope. Several inner membrane-containing viruses have been described for archaea, and their lipid acquisition models are reminiscent to those of PM2 and PRD1. Unselective acquisition of lipids has been observed for bacterial mycoplasmaviruses and archaeal pleolipoviruses, which resemble each other by size, morphology, and life style. In addition to these shared morphotypes of bacterial and archaeal viruses, archaea are infected by viruses with unique morphotypes, such as lemon-shaped, helical, and globular ones. It appears that structurally related viruses may or may not have a lipid component in the virion, suggesting that the significance of viral lipids might be to provide viruses extended means to interact with the host cell.

System analysis of synonymous codon usage biases in archaeal virus genomes.

Recent studies of geothermally heated aquatic ecosystems have found widely divergent viruses with unusual morphotypes. Archaeal viruses isolated from these hot habitats usually have double-stranded DNA genomes, linear or circular, and can infect members of the Archaea domain. In this study, the synonymous codon usage bias (SCUB) and dinucleotide composition in the available complete archaeal virus genome sequences have been investigated. It was found that there is a significant variation in SCUB among different Archaeal virus species, which is mainly determined by the base composition. The outcome of correspondence analysis (COA) and Spearman׳s rank correlation analysis shows that codon usage of selected archaeal virus genes depends mainly on GC richness of genome, and the gene׳s function, albeit with smaller effects, also contributes to codon usage in this virus. Furthermore, this investigation reveals that aromaticity of each protein is also critical in affecting SCUB of these viral genes although it was less important than that of the mutational bias. Especially, mutational pressure may influence SCUB in SIRV1, SIRV2, ARV1, AFV1, and PhiCh1 viruses, whereas translational selection could play a leading role in HRPV1׳s SCUB. These conclusions not only can offer an insight into the codon usage biases of archaeal virus and subsequently the possible relationship between archaeal viruses and their host, but also may help in understanding the evolution of archaeal viruses and their gene classification, and more helpful to explore the origin of life and the evolution of biology.

A novel single-tailed fusiform Sulfolobus virus STSV2 infecting model Sulfolobus species.

A newly isolated single-tailed fusiform virus, Sulfolobus tengchongensis spindle-shaped virus STSV2, from Hamazui, China, is characterised. It contains a double-stranded modified DNA genome of 76,107 bp and is enveloped by a lipid membrane structure. Virions exhibit a single coat protein that forms oligomers when isolated. STSV2 is related to the single-tailed fusiform virus STSV1 and, more distantly, to the two-tailed bicaudavirus ATV. The virus can be stably cultured over long periods in laboratory strains of Sulfolobus and no evidence was found for cell lysis under different stress conditions. Therefore, it constitutes an excellent model virus for archaeal virus-host studies.

Host and viral transcriptional regulators in Sulfolobus: an overview.

The genus Sulfolobus includes microorganisms belonging to the domain Archaea, sub-kingdom Crenarchaeota, living in geographically distant acidic hot springs. Their adaptation to such particular habitats requires finely regulated mechanisms of gene expression, among which, those modulated by sequence-specific transcription factors (TFs) play a key role. In this review, we summarize the current knowledge on the repertoires of TFs found in Sulfolobus spp. and their viruses, focusing on the description of their DNA-binding domains and their structure-function relationship.

DNA ejection from an archaeal virus--a single-molecule approach.

The translocation of genetic material from the viral capsid to the cell is an essential part of the viral infection process. Whether the energetics of this process is driven by the energy stored within the confined nucleic acid or cellular processes pull the genome into the cell has been the subject of discussion. However, in vitro studies of genome ejection have been limited to a few head-tailed bacteriophages with a double-stranded DNA genome. Here we describe a DNA release system that operates in an archaeal virus. This virus infects an archaeon Haloarcula hispanica that was isolated from a hypersaline environment. The DNA-ejection velocity of His1, determined by single-molecule experiments, is comparable to that of bacterial viruses. We found that the ejection process is modulated by the external osmotic pressure (polyethylene glycol (PEG)) and by increased ion (Mg(2+) and Na(+)) concentration. The observed ejection was unidirectional, randomly paused, and incomplete, which suggests that cellular processes are required to complete the DNA transfer.