The crystal structure of a virus-like particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses

Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).     

Does common architecture reveal a viral lineage spanning all three domains of life?

Our discovery that the major coat protein of bacteriophage PRD1 resembles that of human adenovirus raised the unexpected possibility that viruses infecting bacteria could be related by evolution to those infecting animal hosts. We first review the development of this idea. We then describe how we have used structure-based modeling to show that several other viruses with no detectable sequence similarity are likely to have coats constructed from similar proteins-the "double-barrel trimer." There is evidence that the group includes a diversity of viruses infecting very different hosts in all three domains of life: Eukarya; Bacteria; and Archaea that diverged billions of years ago. The current classification of viruses obscures such similarities. We propose that the occurrence of a double-barrel trimer coat protein in an icosahedral dsDNA virus with large facets, irrespective of its host, is a very strong indicator of its membership in a lineage of viruses with a common ancestor.     

Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.     

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.     

The Architecture and Chemical Stability of the Archaeal Sulfolobus Turreted Icosahedral Virus

Viruses utilize a diverse array of mechanisms to deliver their genomes into hosts. While great strides have been made in understanding the genome delivery of eukaryotic and prokaryotic viruses, little is known about archaeal virus genome delivery and the associated particle changes. The Sulfolobus turreted icosahedral virus (STIV) is a double-stranded DNA (dsDNA) archaeal virus that contains a host-derived membrane sandwiched between the genome and the proteinaceous capsid shell. Using cryo-electron microscopy (cryo-EM) and different biochemical treatments, we identified three viral morphologies that may correspond to biochemical disassembly states of STIV. One of these morphologies was subtly different from the previously published 27-Å-resolution electron density that was interpreted with the crystal structure of the major capsid protein (MCP). However, these particles could be analyzed at 12.5-Å resolution by cryo-EM. Comparing these two structures, we identified the location of multiple proteins forming the large turret-like appendages at the icosahedral vertices, observed heterogeneous glycosylation of the capsid shell, and identified mobile MCP C-terminal arms responsible for tethering and releasing the underlying viral membrane to and from the capsid shell. Collectively, our studies allow us to propose a fusogenic mechanism of genome delivery by STIV, in which the dismantled capsid shell allows for the fusion of the viral and host membranes and the internalization of the viral genome.Viruses are valuable biological tools for manipulating the cellular processes of their hosts, and they can also serve as model systems for describing macromolecular interactions through the analysis of their architecture. The Sulfolobus turreted icosahedral virus (STIV) is an archaeal virus that infects Sulfolobus solfataricus (phylum Crenarchaeota). STIV is a lytic virus that was isolated from an acidic hot spring (>80°C and pH of <3) in Yellowstone National Park (27). Hence, STIV is an important model for studying the biochemical requirements to sustain life in extreme physicochemical conditions and has the potential to become a tool for the biochemical and genetic manipulation of its host—much like bacteriophages lambda, P22, and phi29 have done for their respective hosts.Prior structural studies of STIV using cryo-electron microscopy (cryo-EM), X-ray crystallography, and proteomics have described large pentameric turret-like structures, with petal-like protrusions emanating from their central shafts (27). The T=31d capsid shell is composed of trimeric capsomers exhibiting pseudo-hexagonal symmetry, in which each of the three capsomer subunits donates two viral jelly rolls with its β-sheets normal to the capsid surface (15, 27). Capsomers surrounding the icosahedral 3-fold axes, and their neighboring subunits, make direct contact with the viral membrane via a highly basic C-terminal helix of each subunit (15, 23). Surrounding the base of the turrets are proteins that make contact with the capsid shell and a host-derived viral membrane (15). The viral membrane and the enclosed viral genome are referred to as the lipid core.The capsid architecture of STIV and the crystal structure of its major capsid protein (MCP) are strikingly similar to those of the bacteriophages PRD1, Bam35, and PM2, the alga virus PBCV-1, and the mammalian adenovirus. This similarity suggests that these viruses share an ancestral virus (2, 4, 7, 15, 25). Given the evolutionary relationship shared between STIV and PRD1, we postulated that the large turret-like vertices of STIV were used to inject the viral genome into the Sulfolobus host—a genome delivery mechanism employed by PRD1 (27).A recent report by Brumfield et al. (5) describes gross cellular ultrastructural changes induced in the Sulfolobus host during STIV infection and release. The authors identified distinct particles that appear to be assembly intermediates of STIV en route to maturation. From these intermediates the authors proposed a general mechanism of capsid assembly, in which MCP subunits and minor capsid proteins (mCPs) coassemble with the lipid membrane to form a lipid-enclosed protein vesicle. These vesicles are spherical and lack the double-stranded DNA (dsDNA) genome and turret-like appendages at the vertices.While these studies confirm an empty procapsid intermediate, the corresponding molecular mechanism associated with assembly and disassembly remains to be understood. Moreover, little is known about STIV or other archaeal virus genome delivery into the host. To obtain a better understanding of the molecular mechanism of STIV architecture and its role in genome delivery, we characterized three distinct morphologies of STIV particles using cryo-EM. An image reconstruction of one of these revealed the absence of a number of constituents decorating the STIV capsid. Hence, for simplicity, we refer to the previously reported image reconstruction (27) as “decorated” and the new image reconstruction reported here as “undecorated.” Reference-free two-dimensional (2D) class averages of the second identified morphology reveal a partially decorated STIV lipid core. The third identified morphology corresponds to the isolated STIV lipid core. Taken together, our analyses indicate that these morphologies correspond to different disassembly intermediates of STIV that can be isolated in vitro and help provide a picture of the STIV capsid architecture. Additionally, these morphologies allow us to propose an alternative possible mechanism of genome delivery.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

Structure of A197 from Sulfolobus turreted icosahedral virus: a crenarchaeal viral glycosyltransferase exhibiting the GT-A fold

Sulfolobus turreted icosahedral virus (STIV) was the first icosahedral virus characterized from an archaeal host. It infects Sulfolobus species that thrive in the acidic hot springs (pH 2.9 to 3.9 and 72 to 92 degrees C) of Yellowstone National Park. The overall capsid architecture and the structure of its major capsid protein are very similar to those of the bacteriophage PRD1 and eukaryotic viruses Paramecium bursaria Chlorella virus 1 and adenovirus, suggesting a viral lineage that predates the three domains of life. The 17,663-base-pair, circular, double-stranded DNA genome contains 36 potential open reading frames, whose sequences generally show little similarity to other genes in the sequence databases. However, functional and evolutionary information may be suggested by a protein's three-dimensional structure. To this end, we have undertaken structural studies of the STIV proteome. Here we report our work on A197, the product of an STIV open reading frame. The structure of A197 reveals a GT-A fold that is common to many members of the glycosyltransferase superfamily. A197 possesses a canonical DXD motif and a putative catalytic base that are hallmarks of this family of enzymes, strongly suggesting a glycosyltransferase activity for A197. Potential roles for the putative glycosyltransferase activity of A197 and their evolutionary implications are discussed.     

Hot crenarchaeal viruses reveal deep evolutionary connections

The discovery of archaeal viruses provides insights into the fundamental biochemistry and evolution of the Archaea. Recent studies have identified a wide diversity of archaeal viruses within the hot springs of Yellowstone National Park and other high-temperature environments worldwide. These viruses are often morphologically unique and code for genes with little similarity to other known genes in the biosphere, a characteristic that has complicated efforts to trace their evolutionary history. Comparative genomics combined with structural analysis indicate that spindle-shaped virus lineages might be unique to the Archaea, whereas other icosahedral viruses might share a common lineage with viruses of Bacteria and Eukarya. These studies provide insights into the evolutionary history of viruses in all three domains of life.     

An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope

Archaeal organisms are generally known as diverse extremophiles, but they play a crucial role also in moderate environments. So far, only about 50 archaeal viruses have been described in some detail. Despite this, unusual viral morphotypes within this group have been reported. Interestingly, all isolated archaeal viruses have a double-stranded DNA (dsDNA) genome. To further characterize the diversity of archaeal viruses, we screened highly saline water samples for archaea and their viruses. Here, we describe a new haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1) that was isolated from a solar saltern and infects an indigenous host belonging to the genus Halorubrum . Infection does not cause cell lysis, but slightly retards growth of the host and results in high replication of the virus. The sequenced genome (7048 nucleotides) of HRPV-1 is single-stranded DNA (ssDNA), which makes HRPV-1 the first characterized archaeal virus that does not have a dsDNA genome. In spite of this, similarities to another archaeal virus were observed. Two major structural proteins were recognized in protein analyses, and by lipid analyses it was shown that the virion contains a membrane. Electron microscopy studies indicate that the enveloped virion is pleomorphic (approximately 44 × 55 nm). HRPV-1 virion may represent commonly used virion architecture, and it seems that structure-based virus lineages may be extended to non-icosahedral viruses.     

Proteomic analysis of Sulfolobus solfataricus during Sulfolobus Turreted Icosahedral Virus infection

Where there is life, there are viruses. The impact of viruses on evolution, global nutrient cycling, and disease has driven research on their cellular and molecular biology. Knowledge exists for a wide range of viruses; however, a major exception are viruses with archaeal hosts. Archaeal virus-host systems are of great interest because they have similarities to both eukaryotic and bacterial systems and often live in extreme environments. Here we report the first proteomics-based experiments on archaeal host response to viral infection. Sulfolobus Turreted Icosahedral Virus (STIV) infection of Sulfolobus solfataricus P2 was studied using 1D and 2D differential gel electrophoresis (DIGE) to measure abundance and redox changes. Cysteine reactivity was measured using novel fluorescent zwitterionic chemical probes that, together with abundance changes, suggest that virus and host are both vying for control of redox status in the cells. Proteins from nearly 50% of the predicted viral open reading frames were found along with a new STIV protein with a homologue in STIV2. This study provides insight to features of viral replication novel to the archaea, makes strong connections to well-described mechanisms used by eukaryotic viruses such as ESCRT-III mediated transport, and emphasizes the complementary nature of different omics approaches.     

Exceptional virion release mechanism: one more surprise from archaeal viruses

Virion release from the host cell is the final and essential step for completion of the viral life cycle and spread of virions in the environment. Although for eukaryotic and bacterial viruses the egress mechanisms are reasonably well understood, this subject has not been studied in detail for archaeal viruses until recently. Here we summarize available data on the extraordinary egress mechanism exploited by the Sulfolobus islandicus rod-shaped virus SIRV2 and the Sulfolobus turreted icosahedral virus STIV. In addition, we describe features of the virus-induced pyramidal formation, VAP, involved in this process. Being an autonomous structure different from the capsid, the VAP can be considered as a representative of a specific class of virus-coded structures which we suggest to name 'virodomes'.     

Giant viruses coexisted with the cellular ancestors and represent a distinct supergroup along with superkingdoms Archaea, Bacteria and Eukarya

ABSTRACT: BACKGROUND: The discovery of giant viruses with genome and physical size comparable to cellular organisms, remnants of protein translation machinery and virus-specific parasites (virophages) have raised intriguing questions about their origin. Evidence advocates for their inclusion into global phylogenomic studies and their consideration as a distinct and ancient form of life. RESULTS: Here we reconstruct phylogenies describing the evolution of proteomes and protein domain structures of cellular organisms and double-stranded DNA viruses with medium-to-very-large proteomes (giant viruses). Trees of proteomes define viruses as a 'fourth supergroup' along with superkingdoms Archaea, Bacteria, and Eukarya. Trees of domains indicate they have evolved via massive and primordial reductive evolutionary processes. The distribution of domain structures suggests giant viruses harbor a significant number of protein domains including those with no cellular representation. The genomic and structural diversity embedded in the viral proteomes is comparable to the cellular proteomes of organisms with parasitic lifestyles. Since viral domains are widespread among cellular species, we propose that viruses mediate gene transfer between cells and crucially enhance biodiversity. CONCLUSIONS: Results call for a change in the way viruses are perceived. They likely represent a distinct form of life that either predated or coexisted with the last universal common ancestor (LUCA) and constitute a very crucial part of our planet's biosphere.     

Potential role of cellular ESCRT proteins in the STIV life cycle

We are examining the archaeal virus STIV (Sulfolobus turreted icosahedral virus) in order to elucidate the details of its replication cycle and its interactions with its cellular host, Sulfolobus solfataricus. Infection of Sulfolobus by STIV initiates an unusual cell lysis pathway. One component of this pathway is the formation of pyramid-like structures on the surface of infected cells. Multiple seven-sided pyramid-like structures are formed on infected cells late in the STIV replication cycle. These pyramid-like structures are formed at sites where the Sulfolobus S-layer has been disrupted and through which the cellular membrane protrudes. It is through the pyramid-like structures that virus-induced cell lysis occurs in the final stages of the STIV replication cycle. The pathway and process by which these unusual lysis structures are produced appears to be novel to archaeal viruses and are not related to the well-characterized lysis mechanisms utilized by bacterial viruses. We are interested in elucidating both the viral and cellular components involved with STIV lysis of its infected cell. In particular, we are examining the potential role that Sulfolobus ESCRT (endosomal sorting complex required for transport)-like proteins play during viral infection and lysis. We hypothesize that STIV takes advantage of the Sulfolobus ESCRT machinery for virus assembly, transport and cellular lysis.     

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.     

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.     

Phylogenetic and phyletic studies of informational genes in genomes highlight existence of a 4 domain of life including giant viruses

The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.     

Genomics and early cellular evolution. The origin of the DNA world.

The sequencing of several genomes from each of the three domains of life (Archaea, Bacteria and Eukarya) has provided a huge amount of data that can be used to gain insight about early cellular evolution. Some features of the universal tree of life based on rRNA polygenies have been confirmed, such as the division of the cellular living world into three domains. The monophyly of each domain is supported by comparative genomics. However, the hyperthermophilic nature of the 'last universal common ancestor' (LUCA) is not confirmed. Comparative genomics has revealed that gene transfers have been (and still are) very frequent in genome evolution. Nevertheless, a core of informational genes appears more resistant to transfer, testifying for a close relationship between archaeal and eukaryal informational processes. This observation can be explained either by a common unique history between Archaea and Eukarya or by an atypical evolution of these systems in Bacteria. At the moment, comparative genomics still does not allow to choose between a simple LUCA, possibly with an RNA genome, or a complex LUCA, with a DNA genome and informational mechanisms similar to those of Archaea and Eukarya. Further comparative studies on informational mechanisms in the three domains should help to resolve this critical question. The role of viruses in the origin and evolution of DNA genomes also appears an area worth of active investigations. I suggest here that DNA and DNA replication mechanisms appeared first in the virus world before being transferred into cellular organisms.     

Lipid droplets and hepatitis C virus infection

Lipid droplets play an important part in the life cycle of hepatitis C virus and also are markers for steatosis, which is a common condition that arises during infection. These storage organelles are targeted by the viral core protein, which forms the capsid shell. Attachment of core to lipid droplets requires a C-terminal domain within the protein that is highly conserved between different virus isolates. In infected cells, the presence of core on lipid droplets creates loci that contain viral RNA and non-structural proteins involved in genome replication. Such locations may represent sites for initiating assembly and production of nascent virions. In addition to utilising lipid droplets as part the virus life cycle, hepatitis C virus induces their accumulation in infected hepatocytes. The mechanisms involved in this process are not understood but evidence from patient-based studies and model systems suggests the involvement of both viral and host factors.