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.

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.

Unique architecture of thermophilic archaeal virus APBV1 and its genome packaging.

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

HCIV-1 and Other Tailless Icosahedral Internal Membrane-Containing Viruses of the Family Sphaerolipoviridae.

Members of the virus family Sphaerolipoviridae include both archaeal viruses and bacteriophages that possess a tailless icosahedral capsid with an internal membrane. The genera Alpha- and Betasphaerolipovirus comprise viruses that infect halophilic euryarchaea, whereas viruses of thermophilic Thermus bacteria belong to the genus Gammasphaerolipovirus. Both sequence-based and structural clustering of the major capsid proteins and ATPases of sphaerolipoviruses yield three distinct clades corresponding to these three genera. Conserved virion architectural principles observed in sphaerolipoviruses suggest that these viruses belong to the PRD1-adenovirus structural lineage. Here we focus on archaeal alphasphaerolipoviruses and their related putative proviruses. The highest sequence similarities among alphasphaerolipoviruses are observed in the core structural elements of their virions: the two major capsid proteins, the major membrane protein, and a putative packaging ATPase. A recently described tailless icosahedral haloarchaeal virus, Haloarcula californiae icosahedral virus 1 (HCIV-1), has a double-stranded DNA genome and an internal membrane lining the capsid. HCIV-1 shares significant similarities with the other tailless icosahedral internal membrane-containing haloarchaeal viruses of the family Sphaerolipoviridae. The proposal to include a new virus species, Haloarcula virus HCIV1, into the genus Alphasphaerolipovirus was submitted to the International Committee on Taxonomy of Viruses (ICTV) in 2016.

A Greasy Aid to Capsid Assembly: Lessons from a Salty Virus.

In this issue of Structure, Gil-Carton et al. (2015) use hybrid structural methods to investigate the architecture of the membrane-containing halovirus HHIV-2, a member of the PRD1-adenovirus lineage. This work sheds light on how lipid-proteins interactions guide the assembly of single ß-barrel coat proteins to form an icosahedral capsid.

Unification of the globally distributed spindle-shaped viruses of the Archaea.

Viruses with spindle-shaped virions are abundant in diverse environments. Over the years, such viruses have been isolated from a wide range of archaeal hosts. Evolutionary relationships between them remained enigmatic, however. Here, using structural proteins as markers, we define familial ties among these "dark horses" of the virosphere and segregate all spindle-shaped viruses into two distinct evolutionary lineages, corresponding to Bicaudaviridae and Fuselloviridae. Our results illuminate the utility of structure-based virus classification and bring additional order to the virosphere.

Virion architecture unifies globally distributed pleolipoviruses infecting halophilic archaea.

Our understanding of the third domain of life, Archaea, has greatly increased since its establishment some 20 years ago. The increasing information on archaea has also brought their viruses into the limelight. Today, about 100 archaeal viruses are known, which is a low number compared to the numbers of characterized bacterial or eukaryotic viruses. Here, we have performed a comparative biological and structural study of seven pleomorphic viruses infecting extremely halophilic archaea. The pleomorphic nature of this novel virion type was established by sedimentation analysis and cryo-electron microscopy. These nonlytic viruses form virions characterized by a lipid vesicle enclosing the genome, without any nucleoproteins. The viral lipids are unselectively acquired from host cell membranes. The virions contain two to three major structural proteins, which either are embedded in the membrane or form spikes distributed randomly on the external membrane surface. Thus, the most important step during virion assembly is most likely the interaction of the membrane proteins with the genome. The interaction can be driven by single-stranded or double-stranded DNA, resulting in the virions having similar architectures but different genome types. Based on our comparative study, these viruses probably form a novel group, which we define as pleolipoviruses.

In vivo assembly of an archaeal virus studied with whole-cell electron cryotomography.

We applied whole-cell electron cryotomography to the archaeon Sulfolobus infected by Sulfolobus turreted icosahedral virus (STIV), which belongs to the PRD1-Adeno lineage of dsDNA viruses. STIV infection induced the formation of pyramid-like protrusions with sharply defined facets on the cell surface. They had a thicker cross-section than the cytoplasmic membrane and did not contain an exterior surface protein layer (S-layer). Intrapyramidal bodies often occupied the volume of the pyramids. Mature virions, procapsids without genome cores, and partially assembled particles were identified, suggesting that the capsid and inner membrane coassemble in the cytoplasm to form a procapsid. A two-class reconstruction using a maximum likelihood algorithm demonstrated that no dramatic capsid transformation occurred upon DNA packaging. Virions tended to form tightly packed clusters or quasicrystalline arrays while procapsids mostly scattered outside or on the edges of the clusters. The study revealed vivid images of STIV assembly, maturation, and particle distribution in cell.

The single-stranded DNA genome of novel archaeal virus halorubrum pleomorphic virus 1 is enclosed in the envelope decorated with glycoprotein spikes.

Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.

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 x 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.

Phage diversity in a methanogenic digester.

It has been shown that phages are present in natural and engineered ecosystems and influence the structure and performance of prokaryotic communities. However, little has been known about phages occurring in anaerobic ecosystems, including those in methanogenic digesters for waste treatment. This study investigated phages produced in an upflow anaerobic sludge blanket methanogenic digester treating brewery wastes. Phage-like particles (PLPs) in the influent and effluent of the digester were concentrated and purified by sequential filtration and quantified and characterized by transmission electron microscopy (TEM), fluorescence assay, and field inversion gel electrophoresis (FIGE). Results indicate that numbers of PLPs in the effluent of the digester exceeded 1 x 10(9) L-1 and at least 10 times greater than those in the influent, suggesting that substantial amounts of PLPs were produced in the digester. A production rate of the PLPs was estimated at least 5.2 x 10(7) PLPs day-1 L-1. TEM and FIGE showed that a variety of phages were produced in the digester, including those affiliated with Siphoviridae, Myoviridae, and Cystoviridae.

Quantitative dissociation of archaeal virus SH1 reveals distinct capsid proteins and a lipid core.

Viruses infecting archaeal cells are less well understood than those infecting eukaryotic and bacterial cells. Here we study the distribution of the structural proteins between the capsid and the membrane of icosahedral SH1 virus, an archaeal virus infecting extreme halophilic Haloarcula hispanica cells. General features such as morphology, linear dsDNA genome and presence of lipids suggest that it may belong to the recently proposed PRD1-adenovirus lineage of viruses. To investigate this we have initiated structural studies of the virion. Quantitative dissociation of SH1 by 3 M urea or by lowering the salt concentration identified a number of soluble capsid-associated proteins (VP2, VP3, VP4, VP6, VP7 and VP9). These released proteins left behind a particle, or lipid core, containing two major proteins VP10 and VP12 and viral phospholipids. VP1 was released from the lipid core in low ionic strength conditions but not with 3 M urea. Approximately half of the protein VP5 stayed with the lipid core and the other half was released. Analysis of the soluble capsid-associated proteins by their sedimentation and hydrodynamic properties suggests that the most abundant proteins, putative capsomers VP4 and VP7, form an intricate pattern of protein complexes. We also observed large differences in the sizes of the complexes determined by the two different methods suggesting an elongated overall structure for most of the capsid-associated proteins or protein complexes. This work verifies that there is an internal membrane vesicle residing inside the complex icosahedral capsid that is akin to the overall structure of PRD1-like viruses.

Do viruses form lineages across different domains of life?

The scarce characterisation of the viral world has hampered our efforts to appreciate the magnitude and diversity of the viral domain. It appears that almost every species can be infected by a number of viruses. As our knowledge of viruses increases, it appears that this myriad of viruses may be organised into a reasonably low number of viral lineages including members infecting hosts belonging to different domains of life. Viruses belonging to a lineage share a common innate "self" that refers to structural and assembly principles of the virion. This hypothesis has a few consequences. All viruses are old, maybe preceding cellular life, and virus origins are polyphyletic, as opposed to the idea of a monophyletic origin of cellular life.