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 Download asset Open asset Cryo-EMs of AFV1. Arrows point to regions where the virions have been demembranated. This leads to a narrower diameter of the virions and a greatly increased flexibility, as seen by the loops in a,b. The loss of the membrane does not necessarily occur over the whole virion, as can be seen in c where a membrane-enveloped region in the center is bracketed by two regions with no membrane. The scale bar (b) is 1,000 Å. https://doi.org/10.7554/eLife.26268.003

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 Download asset Open asset The distribution of segments sorted against references containing 1-start helices with different pitch values. The validity of this sorting was confirmed by taking power spectra from different bins, which behaved as expected and showed the helical pitch of the corresponding reference. The reconstruction was generated using segments from the central three bins. https://doi.org/10.7554/eLife.26268.004

Figure 3 Download asset Open asset Three-dimensional reconstruction of AFV1. (a) A slice perpendicular to the filament axis. The red arrows define a distance of 20 Å, the approximate thickness of the membrane enveloping the virions. The membrane has a denser outer component and a less dense inner part, separated by a region of lower density. (b) A view of the protein core, looking from the membrane. The asymmetric unit in the virus is a pseudo-symmetric heterodimer of MCP1 (red) and MCP2 (yellow). (c) A view looking down the filament axis (perpendicular to that in b) with the model for the DNA phosphodiester backbone underneath the protein in blue. The helix-turn helix motif of each subunit faces into the narrow lumen. The resolution is good enough in this region that some bulky amino acids can be unambiguously located, and three Tyr21 residues are labeled. (d) The heterodimer in AFV1 has a pseudo-2-fold symmetry, in contrast to the homodimer in SIRV2 (e) which has strict 2-fold symmetry. In both, A-form DNA is bound within the central cleft. The N- and C-terminal ends in both (d) and (e) are labeled Nt and Ct, respectively. https://doi.org/10.7554/eLife.26268.005

Figure 4 Download asset Open asset (a) An FSC curve between two reconstructions from completely independent sets of segments (having no overlap), each started independently from a full reconstruction filtered to 7 Å resolution. The FSC falls to 0.143 at 1/ (4.1 Å). https://doi.org/10.7554/eLife.26268.006

Figure 5 Download asset Open asset Packing of the DNA in the virion. (a) The phosphate backbone of the DNA model can be fit nicely into the density map, as most of the phosphate groups are well resolved. (b) A slice perpendicular to the DNA axis through the map and model. As expected for A-form DNA (and in contrast to B-form), a hole is seen along the DNA axis, with the bases surrounding this cavity. (c) The Coulombic potential for the AFV1 capsid heterodimer shows significant positive regions (blue) surrounding the DNA, with negative regions (red) away from the DNA. https://doi.org/10.7554/eLife.26268.007

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).

Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Conformational dynamics of 20 lipids from the simulated AFV1 envelope. Rendered in this movie are 20 sequential lipids from the simulated AFV1 envelope in stick form and the capsid protein in cartoon form. Frames are at nanosecond intervals. https://doi.org/10.7554/eLife.26268.008

Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Conformational dynamics of AFV1 envelope lipids and interfacial water. Rendered in this movie are the simulated AFV1 envelope in stick form, all water molecules within 10 Å, and the capsid protein in cartoon form. Frames are at nanosecond intervals. https://doi.org/10.7554/eLife.26268.009

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 Download asset Open asset Lipid distribution of virions different from host cells. (a) Chemical diagrams for the lipids found in Acidianus hospitalis and AFV1. (b) The distribution of lipids in the host membrane (red) differs significantly from the distribution found for AFV1 (blue). The scale is in percentage. https://doi.org/10.7554/eLife.26268.010

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).

Figure 7 with 2 supplements with 2 supplements see all Download asset Open asset Modeling the viral membrane. (a) The cylindrically averaged density profile from EM (blue curve) is well fit by a cylindrical envelope (green curve) of phosphoinositol-GDGT0 lipids in horseshoe conformations (b) with 60% having headgroups facing away from the capsid and 40% having headgroups facing towards the capsid. Molecular dynamics simulations of the protein capsid and phosphoinositol-GDGT0 lipids constructed in this arrangement produced a stable envelope rich in horseshoe-conformation lipids (c), while all other envelope arrangements tested failed to maintain the experimentally-derived thickness of 20–25 Å. The density peak at ~30 Å radius (a) arises from the DNA. The central cryo-EM density (radius <15 Å) could not be explained by the capsid proteins, and most likely involves either a minor viral protein or a host protein (Figure 7—figure supplement 1). Since the symmetry of the virion was imposed on this density, which likely does not have such a symmetry, the density was uninterpretable and removed from the other figures. Reconstructed density profiles from the simulations are shown in Figure 7—figure supplement 2, accompanied by movies of 20 envelope lipids in Video 1 and of the entire envelope and interfacial water layers in Video 2. https://doi.org/10.7554/eLife.26268.011

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.