Virtually every environment on the planet is home to some form of life, even places that, at first glance, appear to be too harsh for any organism to survive in. For example, a microscopic organism known as Acidianus hospitalis thrives in highly acidic environments that are hotter than 80°C, conditions that would kill humans and many other species.

Acidianus hospitalis has many adaptations that allow it to survive in its extreme environment. For example, the membrane that surrounds its cells has a different structure to the membranes that surround the cells of most other species. Membranes are made of molecules known as lipids. Generally these lipids assemble into two distinct layers (known as a bilayer) to form the membrane. However, in A. hospitalis the membrane contains only a single layer of lipids that resembles a bilayer in which lipids in opposite layers have fused together to make longer molecules.

A virus known as AFV1 is able to infect A. hospitalis. Like many other viruses, AFV1 steals part of its host cell’s membrane when it leaves the cell in search of new cells to infect. This stolen membrane helps to protect the virus from its surroundings, however, the structure of the membrane surrounding AFV1 was not known.

Kasson et al. combined a technique called cryo-electron microscopy with computer simulations to study the membrane surrounding AFV1. The study shows that this membrane is only half as thick as the membrane that surrounds A. hospitalis. To make this thinner membrane, flexible lipid molecules from the A. hospitalis membrane bend into a U-shape.

These findings reveal a new type of membrane structure not previously seen in the natural world. In the future, this thinner membrane could have many uses in biotechnology, such as to make probes for medical imaging in patients or to deliver drugs to specific sites in the body. Enveloped by this unusual membrane, these structures may be more resistant to the normal processes that degrade and destroy foreign materials in humans and other organisms.

Many viruses, including some of the most devastating human pathogens such as Ebola virus, are enveloped with a lipid membrane. The membrane is considered to be an adaptation to the host that has been convergently acquired in different virus orders (Buchmann and Holmes, 2015). Some groups of evolutionarily related viruses contain both enveloped and non-enveloped members. One example is provided by hyperthermophilic archaeal viruses of the order Ligamenvirales (Prangishvili and Krupovic, 2012) which contain non-enveloped, rigid rod-shaped viruses of the family Rudiviridae and enveloped, flexible filamentous viruses of the family Lipothrixviridae. Viruses from these two families have many homologous genes and build their virions using structurally similar major capsid proteins. The ~4 Å-resolution cryo-EM structure (DiMaio et al., 2015) of a less-complex rudivirus, the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) (Prangishvili et al., 1999), has shown that the viral double-stranded (ds) DNA is completely insulated from the external medium by the single capsid protein which transforms the viral DNA into A-form, explaining the stability of rudiviruses in extremely aggressive (80°C, pH 3) natural habitats. Virions of lipothrixviruses are more complex and are built from two paralogous major capsid proteins (MCP1 and MCP2) which bind dsDNA to form the nucleocapsid (Goulet et al., 2009). The nucleocapsid is enveloped by a lipid membrane and the termini of the virion are decorated with specialized structures involved in host recognition (Bettstetter et al., 2003).

Membranes of hyperthermophilic archaea often consist of unusual tetraether lipids, which form monolayers rather than bilayers typical of bacterial and eukaryotic membranes (De Rosa and Gambacorta, 1988; Valentine, 2007). It has been demonstrated that such membranes are more rigid and stable than typical bilayers, a property with considerable biotechnological appeal. However, molecular details of membrane remodeling in archaea remain grossly understudied, and how a virus captures a non-bilayer membrane from its host is virtually unknown and is conceptually difficult to reconcile with our current understanding of membrane remodeling by viruses infecting eukaryotes (Harrison, 2008, 2015; Hurley and Hanson, 2010). Comparison of the organization of rudiviruses and lipothrixviruses provides an opportunity to probe structural changes permitted by envelope acquisition and can also explain how the lipid envelope may contribute to resisting stresses posed by the extreme geothermal environment.

To better understand the evolutionary relationship between enveloped and non-enveloped viruses, we used cryo-EM (Figure 1) to determine the structure of Acidianus filamentous virus 1 (AFV1), the prototypical lipothrixvirus infecting the hyperthermophilic and acidophilic archaeon Acidianus hospitalis (Bettstetter et al., 2003), and compared the resultant structure to that of SIRV2.

Figure 1

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Determining the structure of AFV1 was complicated by the fact that the virions are significantly more flexible, both with respect to bending as well as extension and compression, than those of SIRV2. Segments could be classified by the pitch of the prominent helix which ranged from 39 to 47 Å (Figure 2), in contrast to the rather fixed pitch of 42 Å in SIRV2 (DiMaio et al., 2015). A three-dimensional reconstruction (Figure 3) of AFV1 not only reveals the gross morphology but also has allowed us to build a full atomic model for both the two MCP subunits and the DNA. While the Fourier Shell Correlation (FSC) is frequently used as the measure of resolution, numerous concerns have been raised about this metric since it is really a measure of self-consistency and not resolution (Subramaniam et al., 2016). Nevertheless, the ‘gold standard’ FSC (Figure 4) yields an overall resolution of 4.1 Å. We think that this is overly optimistic, and may arise from strong features in the DNA (Figure 5) (given the higher MW of the phosphates, the contrast is greater than for protein). A reasonable estimate (based upon comparison with the atomic model) is ~4.5 Å, but it is clear that parts of the complex (such as the outer helices facing the membrane, Figure 3b) are at a worse resolution, while other parts (such as the helix-turn-helix motifs on the very inside, Figure 3c) are at a better resolution.

Figure 2

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Figure 3

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Figure 4

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Figure 5

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The outer diameter of the virion is ~185 Å, while the diameter of the nucleoprotein core alone (excluding the membrane) is ~135 Å. Surprisingly, the membrane is only ~20–25 Å thick, compared to ~40 Å found for archaeal tetraether monolayer (Valentine, 2007) membranes (Chong et al., 2003; Chugunov et al., 2014) and the 40–60 Å found for other cellular membranes and the viral envelopes derived from them (Hollinshead et al., 1999), but a crude calculation done by integrating the cryo-EM density (which corresponds to the Coulombic potential but will be roughly proportional to mass) suggests that ~40% of the total mass of the virion is due to the membrane. The buoyant density for the AFV1 virions was previously determined using a CsCl gradient (Bettstetter et al., 2003), and it was consistent with other membrane-enveloped dsDNA viruses (King et al., 2011). The helically-averaged membrane shows two clear density peaks, with the highest one on the outside and a lower one at inner radius (Figure 3a). This can be seen more easily in the cylindrically-averaged density distribution of the virion, which yields the radial mass distribution (Figure 7a).

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It is unlikely that any details of the membrane structure are lost due to the fact that either the helical symmetry of the nucleocapsid has been imposed upon the membrane (Figure 3a) or the membrane has been cylindrically averaged (Figure 7a) since the membrane is most likely a two-dimensional fluid. If there were some fine structure in the membrane (e.g., a liquid-crystalline phase with a spacing of ~5 Å) then this would be lost by the helical symmetrization (lost as well, of course, by cylindrical averaging) but would appear in averaged power spectra. Since the membrane accounts for ~40% of the mass of the virions, and since there are no features in the averaged power spectra arising from any liquid crystalline features of the membrane, all evidence suggests that it is fluid. What we have been able to observe is that if we compare the membrane with helical symmetrization with a membrane generated by cylindrically-averaging the helical density, or with that obtained from an ab initio cylindrical symmetry reconstruction (see Materials and methods), we see no systematic differences. This excludes the possibility that the membrane is deformed locally by the protein in a way that the membrane would deviate from cylindrical symmetry by the presence of certain amino acid residues either facing the membrane or inserted into the membrane.

In SIRV2, the radius of the DNA is ~60 Å, while in AFV1 the supercoiling is tighter and the radius is ~30 Å. The twist of the A-form DNA in SIRV2 is 11.2 bp/turn (DiMaio et al., 2015) while in AFV1 it is 10.8 bp/turn: in 10 right-handed turns of the 43 Å pitch AFV1 helix, there are 93 repeats of the DNA, each with 12 bp, so there are 1116 bp per 103 (=93 + 10) right handed turns. These two values (11.2 and 10.8) bracket the ‘canonical’ value of 11 bp/turn frequently given for A-DNA (Vargason et al., 2001). Interestingly, the helical pitch in both SIRV2 and AFV1 is ~42–43 Å, but in SIRV2 there are 14.7 homodimers per turn, while in AFV1 there are only 9.3 heterodimers. It is this looser packing in AFV1 that appears responsible for the greater flexibility and disorder. It also explains why the two viruses with linear dsDNA genomes of very different sizes – 35,450 kb for SIRV2 (Peng et al., 2001) and 20,080 for AFV1 (Bettstetter et al., 2003) – have virions of approximately the same length (about 900 nm). In SIRV2 there are tight contacts across the helical turns, while in AFV1 such contacts are absent (Figure 3b), allowing the virions to bend, extend, and compress. At the same time, due to looser protein packing, the AFV1 genome is not completely covered by the protein, while it is in SIRV2. Consequently, the lipid envelope provides a necessary protection to the AFV1 genome in the highly acidic environment of the natural habitat, rationalizing the presence of the envelope in lipothrixviruses. When the membrane is removed (we assume as an artifact of specimen preparation) the virions become much more flexible (Figure 1). Since the membrane, which has fluid-like properties, is unlikely to be directly responsible for the increased rigidity of the enveloped virions, it suggests that the presence of the membrane constrains the protein and thus indirectly imparts rigidity to the structure.

It was originally proposed from a crystallographic study that the two capsid proteins MCP1 and MCP2 would be packed very differently in the virion (Goulet et al., 2009). A model, based upon crystal structures of most of one capsid protein and a fragment of the second one, proposed that one of the capsid proteins formed an inside core of the virion, with DNA wrapping around it, while the other subunit was on the outside of the DNA and partially inserted into the membrane. Surprisingly, we find that the two capsid proteins form a pseudo-symmetric heterodimer (Figure 3d) that resembles in many ways the symmetric homodimer found in SIRV2 (Figure 3e), and that both interact with the DNA in an equivalent manner. We have accounted for all of the amino acids in the two capsid proteins with the exception of 5 N-terminal residues in both MCP1 and MCP2. However, these residues would be too far from the membrane to contact it. Further, we see no density extending from the protein to the membrane.

There are two main differences between the AFV1 and SIRV2 dimers: (1) In SIRV2 the N-terminal tail forms a long helix with a kink that allows it to continuously wrap around the DNA (Figure 3e), while in AFV1 the N-terminal region of both MCP1 and MCP2 form helix-turn-helix motifs which fold back to cover the DNA on both sides (Figure 3d); (2) In SIRV2 the 2-fold axis of the dimer is perpendicular to the helical axis (and goes through the 2-fold axis in the DNA), generating an overall bipolar symmetry for the virion, while in AFV1 the pseudo-2-fold axis of the heterodimer is tilted by 25.7° and does not intersect the helical axis, so that the virion has an overall polarity visible at fairly low resolution. Details of the wrapping of the A-form DNA by the heterodimer are shown in Figure 5, where it can be seen that a positive Coulombic potential would surround the negatively-charged phosphate backbones of the DNA.

The fact that the membrane is only 20–25 Å thick, half of regular lipid membranes, has led us to investigate the membrane further. Since the membrane lipids would not be synthesized by the virus but must come from the host, we first compared the distribution of lipids (Figure 6a) found in the host with those found in the virion membrane (Figure 6b). There is a striking difference in the distributions showing that the incorporation of the glycerol dibiphytanyl glycerol tetraether (GDGT) lipids from the host is highly selective. While the single most dominant species in the host is GDGT-4 (containing 4 cyclopentane moieties), in the virion it is GDGT-0 (containing no cyclopentane moieties), found as only a few percent of the total host membrane lipids. Nevertheless, GDGT-0 is actually one of the most common archaeal membrane lipids (Schouten et al., 2013; Villanueva et al., 2014). Furthermore, it is generally the dominant, or one of the dominant, archaeal lipids in environmental samples taken from soils, rivers, lakes and oceans accounting for >40% of all GDGTs detected (Schouten et al., 2013). The selective incorporation of host lipids in a viral membrane has previously been described, for example, in influenza budding from mammalian cells (Gerl et al., 2012), or in a virus budding from algae (Maat et al., 2014). Such selective incorporation could be driven by direct lipid binding by capsid proteins, enrichment of certain lipid species at sites of viral budding, or physical properties of the viral envelope that cause partitioning of lipids into or out of the nascent envelope during viral budding. Because GDGT-0 is more flexible than the cyclopentane-containing GDGT lipids (Schwarzmann et al., 2015), it can better adopt the horseshoe conformations that have a lower free energy in the highly curved AFV-1 envelope (Galimzyanov et al., 2016). We therefore hypothesize that selective partitioning of GDGT lipids due to the curvature of the envelope is the mechanism for GDGT-0 enrichment in the AFV-1 membrane.

Figure 6

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Knowing the lipid composition of the virion, we used molecular dynamics (MD) to model the viral membrane. Multiple simulations were performed of GDGT-0 lipids arranged cylindrically around the capsid assembly in different densities and orientations. Lipids were modeled with a single phosphoinositol headgroup, as this is the smallest headgroup commonly found on GDGT lipids in the A. hospitalis host (the others are dihexose and sulfonated trihexose headgroups). These lipids frequently adopted a U-shaped or horseshoe conformation, and these horseshoe-rich envelopes with a mix of ‘inward-facing’ and ‘outward-facing’ lipids were the only ones that stably maintained the curvature and thickness observed in the radial density profile from cryo-EM. These lipids still form a monolayer one lipid thick, but the lipids in the monolayer have a mixed orientation. A horseshoe lipid conformation from simulation was therefore used to fit the cryo-EM radial density profile; the best-fit arrangement features 40% of lipids with headgroups facing inwards towards the nucleocapsid and 60% of lipids with headgroups facing towards the outside (Figure 7). Structural models simulated with this lipid orientation maintained a stable envelope structure with the thickness, curvature, propensity to horseshoe conformations, and the slight ~8 Å water-filled gap between envelope and capsid observed by cryo-EM, similar to a surface-supported membrane. The density in this gap observed by cryo-EM was the same as the solvent outside the virus, further suggesting that the region between the envelope and the polar capsid surface and the envelope is similar to that between a supported lipid bilayer and its planar support (Ajo-Franklin et al., 2005; Koenig et al., 1996).

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Simulations very robustly reproduced the width and placement of the envelope density compared to the cryo-EM data, and the density was stable over the course of multiple independent simulations. However, the lipids in the simulations were somewhat more disordered than suggested by the cryo-EM density, such that the double-peak density profile from cryo-EM was smoothed into a broader single peak, and most but not all simulated lipids were in horseshoe conformation. This could result from one of three factors: (1) a slight mismatch in the estimated density of lipids in the viral envelope leading to lateral pressure stresses in the envelope, (2) a larger lipid headgroup present in AFV-1 envelopes than those used in simulations—the simulations used a phosphoinositol headgroup and glycerol backbone as the minimal headgroups found on host lipids, but larger headgroups are also possible, or (3) factors internal to the simulation such as insufficient sampling time or slight mismatches in lipid parameterization. Despite this minor disordering of the lipid tails, the simulated AFV-1 envelope stably maintained a thickness consistent with a single horseshoe-conformation lipid with a thin layer of water between the capsid and envelope. Control simulations that used either incorrect lipid density or single-orientation lipids rather than an ‘in/out mix’ of headgroup orientations did not maintain these features over equivalent simulation timescales. These findings are thus robust and highly consistent with the experimental data.

The simulation models therefore suggest that a mixture of inward-facing and outward-facing lipids primarily in horseshoe conformations is physically stable surrounding a highly curved polar capsid. These simulations match the thickness of the envelope in the electron-density profile and well explain the gap between capsid and envelope as a water layer, but they are not sufficiently powered to distinguish between some-horseshoe and all-horseshoe conformational distributions due to slow conformational relaxations of the lipids and initial-value sensitivity. We have tested sufficient initial conditions to say with confidence that (1) a canonical ‘straight’ tetraether lipid conformation is not compatible with the cylindrical curvature of the capsid; (2) an in/out orientational mix is necessary to capture the gap in density between the capsid and the envelope; and (3) multiple starting conditions with in/out horseshoe start states all produce a stable envelope with a thickness matching that observed experimentally.

These simulations thus provide a specific structural model for the lipids to fit our experimental findings that GDGT-0 lipids in the envelope must occupy a horseshoe conformation based on the cryo-EM density profile of the envelope. The models also predicted that the membrane would account for ~43% of the total mass of the virion, in excellent agreement with the ~40% estimate from the cryo-EM density integration.

AFV-1 is striking because its envelope differs substantially in composition and structure from those of previously described viruses. Because we have combined multiple experimental approaches with computational modeling to analyze the AFV-1 envelope, we briefly recapitulate the lines of evidence for each major finding before going on to discuss some of the important implications of this previously unappreciated envelope structure. Our cryo-EM density data show an envelope surrounding the viral capsid that is ~20 Å in thickness; our mass spectrometry data show that this envelope is composed predominantly of lipids with a GDGT-0 core. The only known conformation of GDGT-0 lipids to form a layer that is <40 Å in thickness is the horseshoe conformation, which has been characterized previously at fluid-air interfaces. Our cryo-EM density data further show an 8 Å region with density corresponding to water between the protein capsid and the lipid, and the structural refinement of the capsid yields a hydrophilic surface. Our computational models then explain these findings via a horseshoe-conformation monolayer with mixed orientation such that (1) GDGT-0 acyl tails are not fully exposed to the external solvent or the polar capsid and (2) the tight curvature of the capsid is well matched by the envelope (which has a radius of curvature on the inside of the membrane of ~70 Å). Thus, the horseshoe-conformation lipid envelope is strongly supported by the experimental data themselves, while the computational model specifies the likely orientation of lipids within this envelope and provides a detailed model of molecular structure.

Despite extensive exploitation of archaeal tetraether lipids for therapeutic purposes, such as archaeosome-based delivery of drugs, cancer vaccines, antigens, genes, etc. (Kaur et al., 2016) remarkably little is known about the actual structure of membranes in Acidianus specifically and Sulfolobales in general. The studies on membranes of Sulfolobales have thus far largely focused on lipid composition in different organisms and on investigation of lipid mixtures in in vitro systems. The lipid composition of Acidianus hospitalis, which we report in this study, is very similar to that previously determined in a related organism, Sulfolobus solfataricus (Quemin et al., 2015) which has a ~5 nm-thick membrane. Interestingly, it has been shown that a spindle-shaped virus SSV1 released from Sulfolobus cells by budding, similar to AFV1, has a membrane enriched in GDGT-0 which is also considerably thinner compared to the host membrane (Quemin et al., 2016). However, this observation remained unexplained. It thus appears that the lipid conformation described in our current study might be more general in enveloped viruses of archaea.

A number of in vitro studies have described archaeal lipids forming U-shaped structures at an air-water interface (Gliozzi et al., 1994; Köhler et al., 2006; Melikyan et al., 1991; Patwardhan and Thompson, 2000; Tomoaia-Cotisel et al., 1992). Since air is extremely hydrophobic, the acyl chains face the air while the polar headgroups of these lipids face the water. The presence of cyclopentane rings (found in the main host species, GDGT-4) has been suggested to rigidify the lipids, making them unable to form a horseshoe (Gliozzi et al., 1994), in agreement with another study which found that more rigid tetraether lipids could not form a horseshoe at the air-water interface while the more flexible lipids did (Patwardhan and Thompson, 2000). Furthermore, theoretical studies suggest that membranes formed from horseshoe conformation lipids have lower curvature energies (Galimzyanov et al., 2016) and would thus be energetically favored on the highly curved AFV-1 surface. This may be the main driving force for the exquisite selectivity seen for the incorporation of host lipids in the viral membrane. A biological role for such a horseshoe conformation has not previously been suggested or found. Our study demonstrates that besides the canonical bacterial/eukaryotic membrane bilayer and archaeal monolayer, there is a third type of biological membrane, the viral horse-shoe membrane layer.

The observation that a membrane that envelops a virus can be formed from lipids in such a conformation opens the door to designing such membranes for applications from drug delivery to nanotechnology. Since the archaeal lipids have been shown to resist phospholipases, extremes of temperature and pH, and can even survive autoclaving, the membrane described here has many potential applications (Patel and Sprott, 1999).

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Author details

1. Peter Kasson

   1. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, United States
   2. Department of Biomedical Engineering, University of Virginia, Charlottesville, United States

   Contribution

   PK, Conceptualization, Software, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-3111-8103

2. Frank DiMaio

   Department of Biochemistry, University of Washington, Seattle, United States

   Contribution

   FD, Software, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0002-7524-8938

3. Xiong Yu

   Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, United States

   Contribution

   XY, Software, Formal analysis, Investigation

   Competing interests

   No competing interests declared.

4. Soizick Lucas-Staat

   Department of Microbiology, Institut Pasteur, Paris, France

   Contribution

   SL-S, Investigation

   Competing interests

   No competing interests declared.

5. Mart Krupovic

   Department of Microbiology, Institut Pasteur, Paris, France

   Contribution

   MK, Investigation

   Competing interests

   No competing interests declared.

   "This ORCID iD identifies the author of this article:" 0000-0001-5486-0098

6. Stefan Schouten

   1. NIOZ Royal Netherlands Institute for Sea Research, Texel, Netherlands
   2. Department of Marine Microbiology and Biogeochemistry, Utrecht University, Texel, Netherlands

   Contribution

   SS, Investigation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared.

7. David Prangishvili

   Department of Microbiology, Institut Pasteur, Paris, France

   Contribution

   DP, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   david.prangishvili@pasteur.fr

   Competing interests

   No competing interests declared.

8. Edward H Egelman

   Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, United States

   Contribution

   EHE, Conceptualization, Funding acquisition, Investigation, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   egelman@virginia.edu

   Competing interests

   EHE: Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-4844-5212

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