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.

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.

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.

Structure and assembly of archaeal viruses.

Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among bacteriophages or viruses of eukaryotes. However, recent environmental studies have shown that archaeal viruses are widespread also in moderate ecosystems, where they play an important ecological role by influencing the turnover of microbial communities, with a global impact on the carbon and nitrogen cycles. In this review, we summarize recent advances in understanding the molecular details of virion organization and assembly of archaeal viruses. We start by briefly introducing the 20 officially recognized families of archaeal viruses and then outline the similarities and differences of archaeal virus assembly with the morphogenesis pathways used by bacterial and eukaryotic viruses, and discuss the evolutionary implications of these observations. Generally, the assembly of the icosahedral archaeal viruses closely follows the mechanisms employed by evolutionarily related bacterial and eukaryotic viruses with the HK97 fold and double jelly-roll major capsid proteins, emphasizing the overall conservation of these pathways over billions of years of evolution. By contrast, archaea-specific viruses employ unique virion assembly mechanisms. We also highlight some of the molecular adaptations underlying the stability of archaeal viruses in extreme environments. Despite considerable progress during the past few years, the archaeal virosphere continues to represent one of the least studied parts of the global virome, with many molecular features awaiting to be discovered and characterized.

The Molecular Mechanism of Cellular Attachment for an Archaeal Virus.

Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus and member of the PRD1-adenovirus lineage. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated studies on STIV attachment and entry. Negative stain and cryoelectron micrographs showed virion attachment to pili-like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub-tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X-ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life.

Survey of high-resolution archaeal virus structures.

Archaeal viruses exhibit diverse morphologies whose structures are just beginning to be explored at high-resolution. In this review, we update recent findings on archaeal structural proteins and virion architectures and place them in the biological context in which these viruses replicate. We conclude that many of the unusual structural features and dynamics of archaeal viruses aid their replication and survival in the chemically harsh environments, in which they replicate. Furthermore, we should expect to find more novel features from examining the high-resolution structures of additional archaeal viruses.

The amazing HK97 fold: versatile results of modest differences.

dsDNA Bacteriophages, some dsDNA archaeal viruses and the Herpesviruses share many features including a common capsid assembly pathway and coat protein fold. The coat proteins of these viruses, which have the HK97 fold, co-assemble with a free or attached scaffolding protein and other capsid proteins into a precursor capsid, known as a procapsid or prohead. The procapsid is a metastable state that increases in stability as a result of morphological changes that occur during the dsDNA packaging reaction. We review evidence from several systems indicating that proper contacts acquired in the assembly of the procapsid are critical to forming the correct morphology in the mature capsid.

Assembly of complex viruses exemplified by a halophilic euryarchaeal virus.

Many of the largest known viruses belong to the PRD1-adeno structural lineage characterised by conserved pseudo-hexameric capsomers composed of three copies of a single major capsid protein (MCP). Here, by high-resolution cryo-EM analysis, we show that a class of archaeal viruses possess hetero-hexameric MCPs which mimic the PRD1-adeno lineage trimer. These hetero-hexamers are built from heterodimers and utilise a jigsaw-puzzle system of pegs and holes, and underlying minor capsid proteins, to assemble the capsid laterally from the 5-fold vertices. At these vertices proteins engage inwards with the internal membrane vesicle whilst 2-fold symmetric horn-like structures protrude outwards. The horns are assembled from repeated globular domains attached to a central spine, presumably facilitating multimeric attachment to the cell receptor. Such viruses may represent precursors of the main PRD1-adeno lineage, similarly engaging cell-receptors via 5-fold spikes and using minor proteins to define particle size.

The structure of a prokaryotic viral envelope protein expands the landscape of membrane fusion proteins.

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.

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.

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

AIMS: Analysis of the stability and safety of Sulfolobus monocaudavirus 1 (SMV1) during passage through the mammalian GI tract. METHODS AND RESULTS: 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 (in vitro) and in mice (in vivo). 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. CONCLUSION: Sulfolobus monocaudavirus 1 appeared stable and safe during passage though the mammalian GI tract. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Large Tailed Spindle Viruses of Archaea: a New Way of Doing Viral Business.

Viruses of Archaea continue to surprise us. Archaeal viruses have revealed new morphologies, protein folds, and gene content. This is especially true for large spindle viruses, which infect only Archaea. We present a comparison of particle morphologies, major coat protein structures, and gene content among the five characterized large spindle viruses to elucidate defining characteristics. Structural similarities and a core set of genes support the grouping of the large spindle viruses into a new superfamily.

Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure.

Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.

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.

Self-assembled bionanoparticles based on the Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) coat protein as a prospective bioscaffold for nanotechnological applications.

Biomolecule-nanoparticle hybrid bioconjugates based on bioscaffolds such as protein cages and virus capsules have been widely studied. Highly stable and durable biotemplates are a vital pillar in constructing bio-inorganic functional hybrid composites. Here, we introduce a highly heat-resistant coat protein (CP) of Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) isolated from the hyperthermophilic archaeon as a prospective biological matrix. Our experiments showed that STSV1 CP was successfully cloned and solubly expressed in the Escherichia coli Rosetta-(DE3) host strain. Protein expression was verified by SDS-PAGE and western blot analysis of the reference C-terminally six-histidine (His6) tagged STSV1 CP (HT-CP). Thermal stability experiments showed that the STSV1 coat protein remained fairly stable at 80 °C. The proteins can be purified facilely by heat treatment followed by size exclusion chromatography (SEC). Transmission electron microscopy (TEM) analysis of the purified STSV1 CP protein aggregates demonstrated that the protein could self-assemble into rotavirus-like nanostructures devoid of genetic materials under our experimental conditions. Similar results were obtained for the HT-CP purified by heat treatment followed by Ni-NTA and SEC, indicating that moderately engineered STSV1 CP can retain its self-assembly property. In addition, the STSV1 CP has a high binding affinity for TiO2 nanoparticles. This illustrates that the STSV1 CP can be used as a bioscaffold in nanobiotechnological applications.

Reversible inactivation and desiccation tolerance of silicified viruses.

Long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. To demonstrate a possible dispersal mechanism, we show that bacteriophage T4, archaeal virus Sulfolobus spindle-shaped virus Kamchatka, and vaccinia virus are reversibly inactivated by mineralization in silica under conditions similar to volcanic hot springs. In contrast, bacteriophage PRD1 is not silicified. Moreover, silicification provides viruses with remarkable desiccation resistance, which could allow extensive aerial dispersal.

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story.

It has been proposed that viruses can be divided into a small number of structure-based viral lineages. One of these lineages is exemplified by bacterial virus Hong Kong 97 (HK97), which represents the head-tailed dsDNA bacteriophages. Seemingly similar viruses also infect archaea. Here we demonstrate using genomic analysis, electron cryomicroscopy, and image reconstruction that the major coat protein fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 has the canonical coat protein fold of HK97. Although it has been anticipated previously, this is physical evidence that bacterial and archaeal head-tailed viruses share a common architectural principle. The HK97-like fold has previously been recognized also in herpesviruses, and this study expands the HK97-like lineage to viruses from all three domains of life. This is only the second established lineage to include archaeal, bacterial, and eukaryotic viruses. Thus, our findings support the hypothesis that the last common universal ancestor of cellular organisms was infected by a number of different viruses.

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.

Crystal structure of PAV1-137: a protein from the virus PAV1 that infects Pyrococcus abyssi.

Pyrococcus abyssi virus 1 (PAV1) was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23) that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137) at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four- α -helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

Modified coat protein forms the flexible spindle-shaped virion of haloarchaeal virus His1.

Extremophiles are found in all three domains of cellular life. However, hyperthermic and hypersaline environments are typically dominated by archaeal cells which also hold the records for the highest growth temperature and are able to grow even at saturated salinity. Hypersaline environments are rich of virus-like particles, and spindle-shaped virions resembling lemons are one of the most abundant virus morphotypes. Spindle-shaped viruses are archaea-specific as all the about 15 such virus isolates infect either hyperthermophilic or halophilic archaea. In the present work, we studied spindle-shaped virus His1 infecting an extremely halophilic euryarchaeon, Haloarcula hispanica. We demonstrate that His1 tolerates a variety of salinities, even lower than that of seawater. The detailed analysis of the structural constituents showed that the His1 virion is composed of only one major and a few minor structural proteins. There is no lipid bilayer in the His1 virion but the major structural protein VP21 is most likely lipid modified. VP21 forms the virion capsid, and the lipid modification probably enables hydrophobic interactions leading to the flexible nature of the virion. Furthermore, we propose that euryarchaeal virus His1 may be related to crenarchaeal fuselloviruses, and that the short-tailed spindle-shaped viruses could form a structure-based viral lineage.