Fig 1.
Cryo-ET of vaccinia-infected cells.
(A) Schematic representation of vaccinia assembly and envelopment. D13-coated membrane crescents close to form a spherical IV (membrane in red), which mature into brick-shaped IMV (membrane in blue) with a viral core that presents 2 concavities, occupied by 2 lateral bodies (black). IMV can be enveloped by a Golgi cisterna or endosomal compartment (orange), resulting in the formation of triple membraned IEV, which can fuse with the plasma membrane to liberate EEV, which are called CEV if they remain attached to the cell surface. (B) Left: Central section of a tomogram showing fully formed IV. The red arrow points at an IV with a lateral invagination, which is detected in 8 out of the 11 IV in the tomogram. Invaginations in other IV can be seen in the 3D-segmented model of the membrane of the same tomogram (right) (see S1 Movie). The sample thickness range was 310–360 nm. (C) Tomogram middle view (left) and its corresponding segmentation (right) of IMV (see S2 Movie). A “cut corner” (magenta arrows) is apparent in some IMV. Sample thickness range was 260–270 nm. Scalebars = 100 nm. CEV, cell-associated enveloped virus; cryo-ET, cryo-electron tomography; EEV, extracellular enveloped virion; IEV, intracellular enveloped virion; IMV, intracellular mature virion; IV, immature virion.
Fig 2.
(A) Tomogram section showing D13-coated membrane crescents with open ends (magenta arrowheads). (B) Tomogram sections of a crescent that is almost closed: The middle view (left) reveals a membrane discontinuity (magenta arrowheads), while in a higher plane (+ 48 nm, right), the membrane is continuous (see S3 Movie). (C) Closed IV containing a condensed nucleoid (outlined in magenta) with repeated structural features (magenta arrowheads). (D) Cytoplasmic condensed nucleoid/s next to an IV. (E) Extended nucleoid/s associated with an open IV. (F) Structured illumination microscopy image of an infected cell showing condensed DNA structures (green) in association with RFP-A3 positive viral particles (magenta) located in a viral factory. Tomogram scalebars = 100 nm. IV, immature virion.
Fig 3.
In situ structure of the D13 lattice.
(A) Tomogram section as indicated in the schematic on the left through the middle and top of a representative IV (see S4 Movie). The densities corresponding to the D13 lattice are coloured in magenta in the right panels and show pseudohexagonal organisation. Scale bar = 100 nm. (B) Map based on STA of D13 and the underlying IV membrane from a top (left) and a side (right) view with fitting of coordinate models of D13 trimers (PDB 7VFE) shown in blue/brown. IV, immature virion; STA, subtomogram averaging.
Fig 4.
Corrugation of the viral membrane during IV to IMV maturation.
(A) Representative IV showing that the viral membrane is smooth. Magnified regions (2 x) are shown to the right, with or without a line tracing the IV membrane. The D13 lattice is marked with an asterisk and arrowheads indicate the viral membrane. (B) A portion (680 × 65 nm) of the IV membrane was segmented and 2 segmentation views are shown illustrating its smoothness. (C) Top left shows a middle section of an IMV, while the bottom left image corresponds to a “side view” of another IMV. In the magnified regions, a cyan line traces the wrinkled IMV membrane (arrowheads). (D) A portion (570–580 × 65 nm) of each viral membrane was segmented and 2 views are shown for each virion. Scale bars = 100 nm. IMV, intracellular mature virion; IV, immature virion.
Fig 5.
(A) Central tomogram section showing the broadest view of a representative IMV together with a region magnified 2.5 times to highlight the IMV layers (see S5 Movie). (B) Lateral view of an IMV showing the lateral bodies (outlined in red) and the two concavities of the core. The magnified regions outlined in magenta show the IMV layers. (C) Different sections of the IMV shown in (A) at the indicated plane positions in nm. The inset in the +31 nm view shows the surface of the palisade lattice. In the second row, yellow highlights the palisade structure, while red indicates the lateral body. (D) The first row shows a schematic representation of the views displayed in (C), while the second row represents the orthogonal view together with the tomogram position (black line). (E) Left: top and side views of a segmentation model of the IMV in (A). The outer layer (blue) and viral membrane (black) are cut away so the internal palisade (yellow) and the lateral bodies (red) can be seen (see S6 Movie). Right: The 2 concavities of the core are more apparent in a Simulated Digitally Reconstructed Radiograph (DDR Rendering) of the same segmentation model. Scale bars = 100 nm. IMV, intracellular mature virion.
Fig 6.
Ultrastructure of the naked viral core.
(A) Middle section of a naked viral core together with a region magnified 3 times to highlight the palisade and inner wall as well as the polymers that surround the core. (B) A higher tomogram section (+41 nm) of the same naked viral core highlighting the pseudohexagonal lattice of the palisade. (C) At an even higher section (+58 nm), ring structures associated with the palisade are evident. The dimensions of the rings (n = 25) from 5 naked cores are indicated together with the standard error of the mean. Scale bars = 100 nm.
Fig 7.
The invariant architecture of the corrugated IMV membrane.
(A) Central plane of tomograms showing that the additional membranes of IEV and CEV (associated with the PM) do not alter the ultrastructure of the internal IMV. A schematic representation illustrating the number of membranes of IMV, IEV, and EEV/CEV is shown beneath the tomograms. (B) Segmented membranes of the CEV shown in (A), highlighting the corrugated character of the inner membrane, which is in contrast to the smooth outer membrane of the CEV. The images on the right show views of the segmented portions of the inner (cyan, 450 × 65 nm) and outer (orange, 520 × 65 nm) membranes. Scale bars = 100 nm. CEV, cell-associated enveloped virus; EEV, extracellular enveloped virion; IEV, intracellular enveloped virion; IMV, intracellular mature virion; PM, plasma membrane.
Fig 8.
The palisade is a pseudohexagonal lattice composed of trimeric proteins.
(A) Sections through the palisade layer corresponding to the virions shown in Fig 7A. Scale bar = 10 nm. (B) Map derived from STA showing the inner wall, palisade layer, viral membrane, and outer layer in surface representation (left) and corresponding sections of the map (right, greyscale). CEV, cell-associated enveloped virus; IEV, intracellular enveloped virion; IMV, intracellular mature virion; STA, subtomogram averaging.
Fig 9.
Maturation of vaccinia virus: From IV to IMV.
(A) Middle view dimensions of IV (diameter, n = 26; perimeter, n = 15) and IMV (major axis, n = 22; intermediate axis, n = 19; minor axis, n = 10; perimeter, n = 19) tomograms. * Indicates that the IMV minor axis was calculated from the side views of 3 IMV and 7 IMV inside EEV (see Methods). For volume calculation, IV and IMV were assumed to be spheres and triaxial ellipsoids, respectively. S3 Table shows the individual numerical data. (B) Plots showing the individual values for perimeter and diameter/major axis (magenta lines in schematics below graph) of the IV, IMV, and EEV/CEV. IV and IMV measurements were used to calculate the averages shown in (A). The averages of the EEV inner-most membrane perimeter and major axis are 1,074.46 nm ± 7.09 nm and 348.91 ± 1.87 nm, respectively. (C) IV and IMV along with possible intermediates together with magnified regions (2.5 times) highlighting the different layers of each particle. Scale bar = 100 nm. (D) Vaccinia maturation model: the palisade lattice (yellow) forms inside D13-coated IV, its maximum length being determined by the IV diameter. Following disassembly of the D13 lattice, the viral membrane acquires the shape of the palisade and becomes wrinkled. During this process, the presence of two lateral bodies (red) results in the deformation of the core, which adopts its characteristic biconcave shape. All errors in (A) and (B) are given as ± standard error of the mean. EEV, extracellular enveloped virion; IMV, intracellular mature virion; IV, immature virion.