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.

Mechanical tomography of an archaeal lemon-shaped virus reveals membrane-like fluidity of the capsid and liquid nucleoprotein cargo.

Archaeal lemon-shaped viruses have unique helical capsids composed of highly hydrophobic protein strands which can slide past each other resulting in remarkable morphological reorganization. Here, using atomic force microscopy, we explore the biomechanical properties of the lemon-shaped virions of Sulfolobus monocaudavirus 1 (SMV1), a double-stranded DNA virus which infects hyperthermophilic (~80 °C) and acidophilic (pH ~ 2) archaea. Our results reveal that SMV1 virions are extremely soft and withstand repeated extensive deformations, reaching remarkable strains of 80% during multiple cycles of consecutive mechanical assaults, yet showing scarce traces of disruption. SMV1 virions can reversibly collapse wall-to-wall, reducing their volume by ~90%. Beyond revealing the exceptional malleability of the SMV1 protein shell, our data also suggest a fluid-like nucleoprotein cargo which can flow inside the capsid, resisting and accommodating mechanical deformations without further alteration. Our experiments suggest a packing fraction of the virus core to be as low as 11%, with the amount of the accessory proteins almost four times exceeding that of the viral genome. Our findings indicate that SMV1 protein capsid displays biomechanical properties of lipid membranes, which is not found among protein capsids of other viruses. The remarkable malleability and fluidity of the SMV1 virions are likely necessary for the structural transformations during the infection and adaptation to extreme environmental conditions.

Revisiting evolutionary trajectories and the organization of the Pleolipoviridae family.

Archaeal pleomorphic viruses belonging to the Pleolipoviridae family represent an enigmatic group as they exhibit unique genomic features and are thought to have evolved through recombination with different archaeal plasmids. However, most of our understanding of the diversity and evolutionary trajectories of this clade comes from a handful of isolated representatives. Here we present 164 new genomes of pleolipoviruses obtained from metagenomic data of Australian hypersaline lakes and publicly available metagenomic data. We perform a comprehensive analysis on the diversity and evolutionary relationships of the newly discovered viruses and previously described pleolipoviruses. We propose to classify the viruses into five genera within the Pleolipoviridae family, with one new genus represented only by virus genomes retrieved in this study. Our data support the current hypothesis that pleolipoviruses reshaped their genomes through recombining with multiple different groups of plasmids, which is reflected in the diversity of their predicted replication strategies. We show that the proposed genus Epsilonpleolipovirus has evolutionary ties to pRN1-like plasmids from Sulfolobus, suggesting that this group could be infecting other archaeal phyla. Interestingly, we observed that the genome size of pleolipoviruses is correlated to the presence or absence of an integrase. Analyses of the host range revealed that all but one virus exhibit an extremely narrow range, and we show that the predicted tertiary structure of the spike protein is strongly associated with the host family, suggesting a specific adaptation to the host S-layer glycoprotein organization.

ICTV Virus Taxonomy Profile: Turriviridae 2024.

The family Turriviridae includes viruses with a dsDNA genome of 16-17 kbp. Virions are spherical with a diameter of approximately 75 nm and comprise a host-derived internal lipid membrane surrounded by a proteinaceous capsid shell. Members of the family Turriviridae infect extremophilic archaea of the genera Sulfolobus and Saccharolobus. Viral infection results in cell lysis for Sulfolobus turreted icosahedral virus 1 infection but other members of the family can be temperate. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Turriviridae, which is available at ictv.global/report/turriviridae.

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.

Diverse viruses of marine archaea discovered using metagenomics.

During the past decade, metagenomics became a method of choice for the discovery of novel viruses. However, host assignment for uncultured viruses remains challenging, especially for archaeal viruses, which are grossly undersampled compared to viruses of bacteria and eukaryotes. Here, we assessed the utility of CRISPR spacer targeting, tRNA gene matching and homology searches for viral signature proteins, such as major capsid proteins, for the assignment of archaeal hosts and validated these approaches on metaviromes from Yangshan Harbor (YSH). We report 35 new genomes of viruses which could be confidently assigned to hosts representing diverse lineages of marine archaea. We show that the archaeal YSH virome is highly diverse, with some viruses enriching the previously described virus groups, such as magroviruses of Marine Group II Archaea (Poseidoniales), and others representing novel groups of marine archaeal viruses. Metagenomic recruitment of Tara Oceans datasets on the YSH viral genomes demonstrated the presence of YSH Poseidoniales and Nitrososphaeria viruses in the global oceans, but also revealed the endemic YSH-specific viral lineages. Furthermore, our results highlight the relationship between the soil and marine thaumarchaeal viruses. We propose three new families within the class Caudoviricetes for the classification of the five complete viral genomes predicted to replicate in marine Poseidoniales and Nitrososphaeria, two ecologically important and widespread archaeal groups. This study illustrates the utility of viral metagenomics in exploring the archaeal virome and provides new insights into the diversity, distribution and evolution of marine archaeal viruses.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments.

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

ICTV Virus Taxonomy Profile: Pleolipoviridae 2022.

Members of the family Pleolipoviridae are pseudo-spherical and pleomorphic archaeal viruses composed of a membrane vesicle, which encloses a DNA genome. The genome is either circular ssDNA or dsDNA, or linear dsDNA molecules of approximately 7 to 17 kilonucleotides or kbp. 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. Some viruses are temperate and can integrate into the host chromosome. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Pleolipoviridae, which is available at ictv.global/report/pleolipoviridae.

Adnaviria: a New Realm for Archaeal Filamentous Viruses with Linear A-Form Double-Stranded DNA Genomes.

The International Committee on Taxonomy of Viruses (ICTV) has recently adopted a comprehensive, hierarchical system of virus taxa. The highest ranks in this hierarchy are realms, each of which is considered monophyletic but apparently originated independently of other realms. Here, we announce the creation of a new realm, Adnaviria, which unifies archaeal filamentous viruses with linear A-form double-stranded DNA genomes and characteristic major capsid proteins unrelated to those encoded by other known viruses.

Egress of archaeal viruses.

Viruses of Archaea, arguably the most mysterious part of the virosphere due to their unique morphotypes and genome contents, exploit diverse mechanisms for releasing virus progeny from the host cell. These include virus release as a result of the enzymatic degradation of the cell wall or budding through it, common for viruses of Bacteria and Eukarya, as well as a unique mechanism of virus egress through small polygonal perforations on the cell surface. The process of the formation of these perforations includes the development of pyramidal structures on the membrane of the infected cell, which gradually grow by the expansion of their faces and eventually open like flower petals. This mechanism of virion release is operating exclusively in cells of hyperthermophilic hosts from the phylum Crenarchaeota, which are encased solely by a layer of surface proteins, S-layer. The review focuses on recent developments in understanding structural and biochemical details of all three types of egress mechanisms of archaeal viruses. TAKE AWAYS: Many archaeal viruses exit the host via polygonal perforations on the cell membrane. The molecular mechanism of exit via specific apertures is unique for archaeal viruses. Some enveloped archaeal viruses exploit the budding mechanism for egress.

A packing for A-form DNA in an icosahedral virus.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

The Flexible Genome of Acidophilic Prokaryotes.

Over the last couple of decades there has been considerable progress in the identification and understanding of the mobile genetic elements that are exchanged between microbes in extremely acidic environments, and of the genes piggybacking on them. Numerous plasmid families, unique viruses of bizarre morphologies and lyfe cycles, as well as plasmid-virus chimeras, have been isolated from acidophiles and characterized to varying degrees. Growing evidence provided by omic-studies have shown that the mobile elements repertoire is not restricted to plasmids and viruses, but that a plethora of integrative elements ranging from miniature inverted repeat transposable elements to large integrative conjugative elements populate the genomes of acidophilic bacteria and archaea. This article reviews the diversity of elements that have been found to constitute the flexible genome of acidophiles. Special emphasis is put on the knowledge generated for Sulfolobus (archaea) and species of the bacterial genera Acidithiobacillus and Leptospirillum. Also, recent knowledge on the strategies used by acidophiles to contain deletereous exchanges while allowing innovation, and the emerging details of the molecular biology of these systems, are discussed. Major lacunae in our understanding of the mobilome of acidophilic prokaryotes and topics for further investigations are identified.

ICTV Virus Taxonomy Profile: Ovaliviridae.

Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae.

ICTV Virus Taxonomy Profile: Thaspiviridae 2021.

Members of the family Thaspiviridae have linear dsDNA genomes of 27 to 29 kbp and are the first viruses known to infect mesophilic ammonia-oxidizing archaea of the phylum Thaumarchaeota. The spindle-shaped virions of Nitrosopumilus spindle-shaped virus 1 possess short tails at one pole and measure 64±3 nm in diameter and 112±6 nm in length. This morphology is similar to that of members of the families Fuselloviridae and Halspiviridae. Virus replication is not lytic but leads to growth inhibition of the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Thaspiviridae, which is available at ictv.global/report/thaspiviridae.

ICTV Virus Taxonomy Profile: Portogloboviridae.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae.

The first head-tailed virus, MFTV1, infecting hyperthermophilic methanogenic deep-sea archaea.

Deep-sea hydrothermal vents are inhabited by complex communities of microbes and their viruses. Despite the importance of viruses in controlling the diversity, adaptation and evolution of their microbial hosts, to date, only eight bacterial and two archaeal viruses isolated from abyssal ecosystems have been described. Thus, our efforts focused on gaining new insights into viruses associated with deep-sea autotrophic archaea. Here, we provide the first evidence of an infection of hyperthermophilic methanogenic archaea by a head-tailed virus, Methanocaldococcus fervens tailed virus 1 (MFTV1). MFTV1 has an isometric head of 50 nm in diameter and a 150 nm-long non-contractile tail. Virions are released continuously without causing a sudden drop in host growth. MFTV1 infects Methanocaldococcus species and is the first hyperthermophilic head-tailed virus described thus far. The viral genome is a double-stranded linear DNA of 31 kb. Interestingly, our results suggest potential strategies adopted by the plasmid pMEFER01, carried by M. fervens, to spread horizontally in hyperthermophilic methanogens. The data presented here open a new window of understanding on how the abyssal mobilome interacts with hyperthermophilic marine archaea.

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.

An Uncultivated Virus Infecting a Nanoarchaeal Parasite in the Hot Springs of Yellowstone National Park.

The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses had been described that infect the Nanoarchaeota. Here, we identify a virus infecting Nanoarchaeota by the use of a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6-kb circular DNA genome coding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single-celled organism that is itself an ectobiont of another single-celled organism.IMPORTANCE Here, we present evidence of the first virus found to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. On the basis of annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family, further demonstrating the remarkable genetic diversity of archaeal viruses.

Novel Sulfolobus Fuselloviruses with Extensive Genomic Variations.

Fuselloviruses are among the most widespread and best-characterized archaeal viruses. They exhibit remarkable diversity, as the list of members of this family is rapidly growing. However, it has yet to be shown how a fuselloviral genome may undergo variation at the levels of both single nucleotides and sequence stretches. Here, we report the isolation and characterization of four novel spindle-shaped viruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22), from a hot spring in the Philippines. SSV19 is a member of the genus Alphafusellovirus, whereas SSV20-22 belong to the genus Betafusellovirus The genomes of SSV20-SSV22 are identical except for the presence of two large variable regions, as well as numerous sites of single-nucleotide polymorphisms (SNPs) unevenly distributed throughout the genomes and enriched in certain regions, including the gene encoding the putative end filament protein VP4. We show that coinfection of the host with SSV20 and SSV22 led to the formation of an SSV21-like virus, presumably through homologous recombination. In addition, large numbers of SNPs were identified in DNA sequences retrieved by PCR amplification targeting the SSV20-22 vp4 gene from the original enrichment culture, indicating the enormous diversity of SSV20-22-like viruses in the environment. The high variability of VP4 is consistent with its potential role in host recognition and binding by the virus.IMPORTANCE How a virus survives in the arms race with its host is an intriguing question. In this study, we isolated and characterized four novel fuselloviruses, named Sulfolobus spindle-shaped viruses 19 to 22 (SSV19-22). Interestingly, SSV20-22 differ primarily in two genomic regions and are apparently convertible through homologous recombination during coinfection. Moreover, sites of single-nucleotide polymorphism (SNP) were identified throughout the genomes of SSV20-22 and, notably, enriched in certain regions, including the gene encoding the putative end filament protein VP4, which is believed to be involved in host recognition and binding by the virus.

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.