Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic "honeycomb" surface coat

Abstract

Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.

Figure 1.

Stages of morphogenesis of vaccinia virus, the prototypical poxvirus, including the abbreviated names of each stage. Virogenesis begins with the formation (from crescent-shaped precursors) of a spherical immature virion (the “IV”). The core of the IV then condenses and differentiates to form a mature intracellular virion (IMV). The naked IMV is then enveloped by a collapsed cisterna of intracellular membrane (of unknown origin) to form an intracellular enveloped virion (the “IEV”). The IEV then behaves like a recycling endosome, moving along microtubules to the cell surface where it ultimately fuses with the plasma membrane to release an IEV derivative that is still wrapped in one of the two layers of its original envelope. Typically, this particle remains attached to the cell after discharge, thus is called a cell-associated enveloped virion or “CEV.” However, if and when it breaks free from the cell surface it is called an extracellular enveloped virion or “EEV” (not depicted). Some CEV's on the cell surface provoke oriented actin polymerization inside the cell (“actin tail formation”), thereby elevating themselves on the tip of a blunt microvillus. This promotes their fusion with neighboring cells and the spread of infection. Bar, 0.3 μm.

Figure 2.

Survey views of viral factories in cells infected overnight and then prepared by different protocols for DEEM. (A) QT6 quail cells infected with the MVA strain of vaccinia virus and quick-frozen alive, without any chemical fixation or other pretreatment. Only immature virions (IVs) are present in this field, but they look different depending on the plane of fracture. Cleanly cross fractured IVs, the large circles with granular interiors, are seen at 1E, 3D, and 5C. Freeze-fractured IVs that have been “scalped” are seen at 3F and 3H (and one with a small “scalping” at 5A). Fractured IVs that have been largely avulsed, leaving only cup-shaped membrane remnants, are seen at 1B, 3B, and 4G. The cross fractured IVs at 1G and 5F show some compaction of their cores and hints of icosahedral shape, indications of incipient maturation. Just peeking out from the uniformly granular cytoplasm at 1C and 4G are two whole unfractured IVs showing surface honeycombs. (B) BS-C-40 cells infected with the WR strain of vaccinia virus and then strongly fixed with glutaraldehyde before freezing. Such fixation coagulates the cytoplasm and causes it to withdraw from all the IVs in the field, thereby providing more expansive views of their honeycombed surfaces (at 3F, 2B, 4A, and 4H). Freeze-fracture views look the same as in Fig. 2 A however, showing both convex “scalped” IVs (at 5B) and concave avulsed IVs (at 3A, 4F). Also present in this field (not depicted in Fig. 2 A) are two incomplete crescent-shaped IV precursors partially filled with viroplasm (at 3B and 4D). (C) Tokuyasu-type cryosection that was freeze dried and replicated rather than viewed in the traditional manner. The preparation for cryosectioning causes even more severe coagulation of the cytoplasm and more exposure of the IV surface honeycomb (at 5B and 4F). Also present in this field are four cross cut intracellular mature viruses (IMVs) at 1B, 2G, 4H, and 5A. The one at 2G is optimally oriented for seeing its internal core. Note that in such cryosections, “scalped” IVs like the one at 4F do not retain any fracture faces. These must break down during cryosectioning and/or thawing. Instead, scalped IVs in such cryosections show complete breaks into their interiors. Bars, 0.3 μm.

Figure 3.

Higher magnifications of cross fractured vaccinia IVs from completely unfixed cells like that depicted in Fig. 2 A. Their surrounding envelopes are invariably >2× thicker than the usual membrane bilayer and are punctuated on their convex surfaces with “pegs” or particles that are presumably the deep-etch equivalent of the “spikes” seen in traditional thin-section views of IVs. A and B display incomplete IV-crescents at different stages of formation. In contrast to their appearance in cells prepared by chemical fixation and dehydration, none of the crescents in quick-frozen cells show any attachments to other cellular membranes, at all. Occasionally their free edges appear to contact nondescript 15–20-nm globules, but otherwise they appear to end completely blindly, as shown here. C illustrates the completed IV sphere. D and E show that other organelles in infected cells that do possess double-thickness envelopes. D shows a mitochondrion whereas E shows a developing vaccinia IEV, where a dark, empty-looking host cell organelle appears to be extending a thin cisternae around the developing virion. In both cases, these organelles cross fracture in a way that clearly shows their construction: two distinct membranes of normal thickness separated by a narrow gap, unlike the IV membrane which is thicker but does not look like two closely opposed membranes. Bars, 0.1 μm.

Figure 4.

Semithin plastic sections of cells infected overnight with MVA, then quick-frozen from life and freeze substituted. Top (3-D): Survey view of a field of IVs with one selected and color coded with membrane in yellow and core in blue. Second (3-D): Survey view of a field of IMVs with one selected and color coded with membrane in yellow and core in two shades of blue, separated by its palisade layer in red. (Top triptych, not 3-D) Higher magnification of portions of IVs showing their single surrounding membrane (yellow) with hints of spikes on their external surfaces. (Middle triptych) Equally high magnifications of portions of IMVs showing the characteristic separation between their outer limiting membrane (yellow) and their palisade layer (red). (Bottom triptych) Portions of three freeze-substituted mitochondria at the same high magnification showing the characteristic thickness of their two membranes and the characteristic separation between them. Bar: (top 3-D views) 0.3 μm; (tryptics) 0.1 μm.

Figure 5.

Direct comparison of the architecture and fracturing properties of the internal “palisade layer” around the cores of intact versus fused virions. (Top) Cross fractures through totally unfixed IMVs from cell like that in Fig. 2 A, showing a clear view of the internal “palisade layers” around their biconcave cores, and showing that these layers do not fracture like a biological lipid membrane. (Bottom) Crossfractures through viral cores recently released into the cytoplasm of a cell exposed to a whopping dose of vaccinia virions by “spinoculation” and quick frozen after only 5 min. The palisade layers surrounding these cores are still clearly discernible and look more or less intact. Nevertheless, even in this naked “exposed' condition in the cytoplasm, they do not freeze fracture like any membrane-containing structure. Instead, they invariably crossfracture like the palisade layers in the intact IMVs above, indicating that they are composed of nothing but protein (and polynucleotides). Bar, 0.1 μm.

Figure 6.

Three different sorts of views of the true surfaces of IMVs. (A) As seen inside whole cells during virogenesis; (B) after natural release as a CEV from an infected cell, followed by rupture of the CEV membrane to expose a clean IMV inside; and (C) after douncing of infected cells and isolation of IMVs as is typically done to harvest virus for subsequent experimentation. Inside of the cell, cytoplasm clings tenaciously to the surface of IMVs, obscuring their surface. (This is apparent also in the survey of Fig. 2 C.) When harvested from cells, most of this obscuring material is removed (C), but the finer features of the IMV surface are degraded into what traditionally have been called “mulberries” (possibly due to the trypsinization and sonication typically used in such purification). When released in the course of a natural infection, and when the outer membranous envelope of the CEV ruptures to expose the IMV to neighboring cells, the IMV surface displays a clear-cut paracrystalline topology defined by parallel double rows of particles (B). Bar, 0.1 μm.

Figure 7.

Direct comparison of clathrin lattices and the honeycomb latices on IVs, shown at exactly the same magnification. The clathrin lattices (top) have lattice parameters >2× greater than the honeycombs on IVs (center). Expansive surface views of IVs are readily obtainable because surrounding cytoplasm appears to be repelled from them, in contradistinction to IMVs (Fig. 6 A). The protein lattice observed on the IV surface is clearly a geodetic honeycomb, and sometimes shows hints of overall icosahedral symmetry. However, its lattice dimensions are much smaller. Its vertex to vertex spacing is 7 ± 1 nm versus clathrin's 15 ± 1 nm. Moreover, the vaccinia lattice typically displays many more lattice-defects and irregularities. These irregularities invariably take the form of “pentamer/heptamer” dislocations, typical for all natural honeycomb lattices. Insets at the bottom illustrate this. Proceeding from the left, green dots indicate proper hexagonal facets and yellow dots indicate inserted pentagons. Only 12 of these pentagonal insertions would be needed in the whole lattice to make it a sphere, according to Crick and Watson's postulate (Crick and Watson, 1956; Caspar and Klug, 1962); but many more pentagons are visible in this relatively small portion of the IV surface. Finally, red dots indicate inserted heptagons and white arrows indicate how each of these heptagons can be matched with a pentagon immediately adjacent to it. Bar, 0.1 μm.

Figure 8.

Freeze fracturing of immature vaccinia virions demonstrates that they are composed of only a single lipid membrane. Panel A diagrams how biological membranes split along the center of the bilayer during freeze fracture, yielding two roughly complementary fracture faces. In the case of vaccinia IVs and IMVs, these fracture faces are of course distinctly convex or concave. Deep etching removes a superficial layer of ice (shaded light in the diagram), thereby exposing the immediately adjacent true surfaces of convexly fractured virions. (B) Gallery of IV convex fractures. Regardless of whether they are chemically fixed before freezing or not, IVs display a single fracture face covered with small protuberances called “intramembrane particles” (which presumably represent transmembrane proteins). Clearly visible on each convex fracture is a step up that marks the transition between the fracture face and the true surface of the IV, where its honeycomb lattice is apparent. (C) Gallery of IV concave fractures. As with the convex fractures above, we have never seen any hint of any jump into what might be a second, closely opposed membrane in the IV envelope. Unlike the convex views, concave fracture faces display only a few intramembrane particles, but mostly randomly oriented fibrils or “stubs” of fibrils, typical of the “plastic distortion” that accompanies freeze fracture even at temperatures below −100°C. Bars, 0.1 μm.

Figure 9.

Freeze-fractured IMVs inside infected cells, illustrating that the fracturing properties of IMVs are basically the same as those of IVs in Fig. 8. Convex fractures are above and concave fractures below. Deep etching is to the left and minimal etching is to the right. (Reduced etching has no effect on the appearance of the fracture faces, but obviously eliminates the glimpses of true surface normally seen around the circumferences of convex fractures. Still, these glimpses look just like the surfaces of the whole IMV seen in Fig. 6 A.) Again, IMVs present only one fracture plane in every instance; hence, they too must be surrounded by only one membrane. Bar, 0.1 μm.

Figure 10.

Diagram comparing how lipid droplets are currently thought to form, versus our hypothesis for poxvirus membrane growth. Lipid droplets are thought to form within the membrane of the ER and then bud from it (top). Here, we propose that the crescent-shaped precursors of vaccinia IVs might acquire phospholipids from the ER membrane in a similar manner (bottom). In both cases, an unstable intermediate in the form of a “T-junction” might form transiently, as is thought to occur during all membrane fusion and budding events.