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Journal of Virology, August 2008, p. 7905-7912, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00194-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Envelope of Intracellular African Swine Fever Virus Is Composed of a Single Lipid Bilayer

Philippa C. Hawes,^1* Christopher L. Netherton,² Thomas E. Wileman,³ and Paul Monaghan¹

Biomaging, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom,¹ Vaccinology Group, Division of Immunology, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom,² Infection and Immunity, School of Medicine, Institute of Health, University of East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom³

Received 28 January 2008/ Accepted 28 May 2008

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African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.

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The assembly and envelopment of the large nucleocytoplasmic DNA viruses such as African swine fever virus (ASFV) and vaccinia virus (VV) have been the subject of many studies, but the mechanisms and pathways that govern the morphogenesis of each virus still remain controversial (1, 11, 12, 23, 24). ASFV virions have a classic icosahedral structure that is similar to that of the iridoviruses, whereas VV immature virions (IV) are spherical. Despite the obvious differences in appearance between intracellular ASFV virions and VV IV, the structural organizations are similar. The center of each virus consists of the genome surrounded by a layer of protein, which in the case of ASFV is called the matrix or inner core shell. This is in turn enclosed by a lipid envelope which lies beneath the capsid (2, 3, 6, 7, 10). The immature VV matures into the brick-shaped intracellular mature virus. ASFV gains a further outer envelope as it buds through the plasma membrane on exit (21). The following experiments refer to the intracellular form of ASFV only.

Considerable analysis of the envelopes of the spherical immature and brick-shaped intracellular mature forms of VV suggests that the envelope originates from membrane crescents formed in the cytoplasm near assembly sites. Early models of VV assembly proposed that the crescents and IV envelopes were single lipid bilayers formed by de novo membrane biogenesis (8). However, sequencing of the poxvirus genome did not support this initial hypothesis, as genes encoding lipid synthesis enzymes were not found (25). In addition, intracellular membranes are usually present as double-membrane cisternae, and it was unclear how the virus could stabilize single bilayers within the cellular environment. In an alternative model, it was suggested that the envelope of the IV was in fact two lipid bilayers closely adhered to each other and that these membranes were derived from cisternae of the endoplasmic reticulum (ER)-Golgi-intermediate compartment of the host cell (24) or, more recently, a domain of the smooth ER (12). Several studies using different transmission electron microscope (TEM) techniques have supported either the single-membrane (15, 16) or double-membrane (11, 12) model. Hollinshead et al. (16) used high-angle tilt-series serial section analysis in the TEM to provide high-resolution images of assembling virus and concluded that inner viral envelopes were formed from a single lipid bilayer and that the second "lipid layer" was actually a protein coat. Recently, it was concluded from deep-etch EM of freeze-fractured, quick-frozen cells that the envelope of the IV is indeed a single lipid bilayer and that the protein coat identified by Hollinshead and others is formed from a continuous honeycomb network of viral D13 protein. Homology between D13 and the capsid proteins of ASFV and the iridoviruses suggests that D13 is the capsid protein of VV (19). Current models suggest that the honeycomb of D13 protein stabilizes the envelope before it closes into a sphere during the formation of the immature virus (15, 27).

ASFV is less well studied than VV but still evokes similar controversy regarding the origin and structure of the intracellular viral envelope. Like for VV, the first description of the morphology of ASFV suggested that the envelope was formed of a single lipid bilayer (2). Biochemical analysis coupled with subcellular fractionation suggested that the viral capsid of ASFV assembled on membranes derived from the ER (4, 5, 6). This was followed up by the work of Rouiller et al. (23), who investigated virus structure by EM of thawed cryosections, which appeared to show that the envelope of ASFV was a double membrane originating from the ER. Andrés et al. (1) proposed a similar model in which assembly of ASFV structural proteins leads to collapse of the ER cisterna and subsequent enwrappment of assembling virions. They concluded that the envelope was derived from a double lipid bilayer but that the two membranes were so tightly apposed during assembly that it was difficult to distinguish between them.

Much of the work investigating the nature of the intracellular viral envelopes of both ASFV and VV has been carried out using the TEM, as the high resolution afforded by this microscope is the clearest way to see virion structure. The experiments have used different methods of preparation, but the most common have been chemical fixation or cryosectioning of infected cells. The two methods produce images with different appearances and therefore lead to differences in interpretation. An alternative method to prepare samples for TEM is high-pressure freezing followed by freeze-substitution. This technique immobilizes samples instantaneously in as near a natural state as is currently possible without the detrimental effects of a chemical fixative (13, 26). In this study we present the first analysis of viral envelopes and cellular membranes in ASFV-infected cells prepared using high-pressure freezing. We compare these results to those obtained from cells prepared by chemical fixation and thawed cryosections and conclude that intracellular virions are enveloped by a single membrane composed of one bilipid layer surrounded by an outer layer formed from the capsid protein. The assembly and envelopment of ASFV therefore appears to follow the same mechanistic principles as for the intracellular mature form of VV.

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Cell culture and virus. Vero (ECACC 84113001) cells, obtained from the European Collection of Animal Cell Cultures, were grown on 13-mm Thermanox plastic coverslips (VWR International Ltd.) before chemical fixation, in 175-ml cell culture flasks before cryosectioning, or 3-mm sapphire glass coverslips (Bal-tec) before high-pressure freezing. Cells were maintained in Dulbecco's modified Eagle medium-HEPES (Sigma) with 10% fetal calf serum (Sigma) at 37°C with 5% carbon dioxide. When the cells reached 80 to 90% confluence, cultures were infected with the ASFV Badajoz 1971 Vero-cell adapted (Ba71v) strain (9) and left for 18 h at 37°C.

TEM of chemically fixed samples. Thermanox coverslips of infected cells were fixed for 1 hour in phosphate-buffered 2% (vol/vol) glutaraldehyde solution before being fixed for a further hour in aqueous 2% (wt/vol) osmium tetroxide at room temperature. Following a dehydration series in ethanol (70% [vol/vol] for 30 min, 90% [vol/vol] for 15 min, and 100% three times for 10 min each), cells on coverslips were washed with propylene oxide (10 min) before being infiltrated with 50% (vol/vol) epoxy resin (Agar Scientific) in propylene oxide for 60 min. After a further 60 min in 100% epoxy resin, coverslips were placed (cell side up) in 18-mm-diameter plastic cups (Taab), covered in fresh epoxy resin, and polymerized at 60°C for 18 h. The plastic cups were removed and coverslips peeled off while the resin was still warm, leaving the cells inside the resin block. Sixty-micrometer sections were cut and grid stained using EMStain (Leica Microsystems) before being imaged in at 100 kV in an FEI Tecnai 12 TEM with a TVIPS F214 digital camera.

Thawed cryosections. Infected-cell monolayers were fixed using phosphate-buffered 2% (wt/vol) glutaraldehyde for 2 h. After a wash in phosphate-buffered saline, 2% (wt/vol) gelatin solution was used to cover the cells while they were gently scraped from the substrate. Cells were spun into a pellet, the supernatant removed, and the pellet resuspended in 12% (wt/vol) gelatin. After another spin, the cell pellet was cooled, removed, and cut into 1-mm² blocks which were infiltrated with 2.3 M sucrose at 4°C overnight. The blocks were attached to cryopins (Leica Microsystems) using 2.3 M sucrose and frozen in liquid nitrogen. After the blocks were trimmed at –90°C in a UCT cryoultramicrotome (Leica Microsystems), 60-nm sections were cut at –120°C, collected on Formvar/carbon-coated copper EM grids, and allowed to reach room temperature. Sections were stained with 3% (wt/vol) aqueous uranyl acetate for 1 minute before being allowed to dry in a thin support film containing 3% (wt/vol) aqueous uranyl acetate and 2% (wt/vol) methyl cellulose at a 1:4 ratio. Samples were imaged in an FEI Tecnai 12 TEM at 100 kV using a TVIPS F214 digital camera.

High-pressure freezing and freeze-substitution. Infected cells on 3-mm sapphire glass coverslips (discs) were frozen and freeze-substituted as described previously (13). Briefly, after freezing in Bal-tec HPM010 HPF, sapphire discs were stored in liquid nitrogen before substitution. During substitution, the sapphire discs were allowed to warm to –90°C, at which time the freeze-substitution medium (2% [wt/vol] uranyl acetate in acetone) was added and left for 1 hour. Infiltration of Lowicryl resin (Agar Scientific) occurred at –50°C, followed by polymerization under UV light at –50°C for 48 h and then at room temperature for a further 48 h. The sapphire discs were removed from the solid resin blocks by immersing them briefly in liquid nitrogen. Thin sections of the resulting resin blocks (60 nm) were cut using Leica Microsystems Ultracut E and imaged without contrasting in an FEI Tecnai 12 TEM at 100 kV using a TVIPS F214 digital camera.

Microscope and camera calibration. System calibration was checked using a standard 2,160-lines/mm carbon diffraction grating (Agar Scientific) in which each line measures 463 nm in width. A number of measurements (n = 20) were taken at a magnification of x30,000 and averaged. The mean and standard deviation of these data (453.17 ± 5.71 nm) indicate that the microscope calibration was accurate to within ±4% at this magnification.

For each of the three treatments, images were collected at the same magnification (x30,000), and where possible, viral envelopes and outer mitochondrial membranes were imaged from the same cell in order to minimize any staining or section thickness irregularities. If it was not possible to take images from the same cell, they were collected from neighboring cells or from cells within the same section. Mitochondria were chosen as the cell membrane compartment to compare to virus because they are large, easily recognizable, and abundant in cells.

Statistical analysis. Numerical values are presented as means ± standard deviations. Measurements of membrane width were collected using digital calipers (RS Components) with an accuracy of 0.03 mm. As the apparent width of a membrane changes with angle, only membranes with crisp outlines were measured and then only at minimum width.

Minitab (v14.13) was used to obtain a normal probability plot to confirm normal distribution of data. Analysis-of-variance tests on (i) virus envelope width means (n = 20) for the three treatments and (ii) outer mitochondrial membrane width means (n = 20) for the three preparation procedures were subsequently carried out. Further analysis of significant differences between pairs of treatments was carried out using Tukey's test (95% confidence intervals). Comparison of mean outer mitochondrial and viral envelope widths within each treatment was carried out using Student's t test (P < 0.05).

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In order to determine the nature of the intracellular viral envelope of ASFV, Vero cells were infected for 18 h with Ba71v and then processed for TEM using either chemical fixation, cryosectioning, or high-pressure freezing. Virions and mitochondria were imaged at a magnification of x30,000 in samples processed by each method. Mitochondria are clearly identifiable cellular organelles with a double membrane and provide a reliable comparison to viral envelopes. In order to negate any processing-induced changes, the ratio of viral envelope to internal control membrane (outer mitochondrial membrane) was calculated for each preparation method.

Figure 1 shows mitochondria and virus prepared by chemical fixation. The five domains of a mitochondrion (Fig. 1a) are the outer mitochondrial membrane, intercristal space, inner mitochondrial membrane, cristae, and matrix. The double membrane of the mitochondrion appears as two dark lines at this magnification (Fig. 1a). However, at high power each lipid leaflet is visible, and therefore each individual membrane appears as two parallel black lines approximately 5 nm apart (Fig. 1e). Because of this, a single membrane, which consists of a lipid bilayer, is often referred to as a "tramline" in the EM. Intracellular ASFV virions from the same section as the mitochondrion shown in Fig. 1A appear as six-sided shapes characteristic of icosahedral viral particles (Fig. 1b). The orientation of virus in the section is crucial to the interpretation of membrane thickness. The virus has to be sectioned such that the membranes are perpendicular to the imaging beam in order for the concentric domains surrounding the matrix to be correctly interpreted. The sides of the virus that have been cut perpendicular to the membrane and those with a nonperpendicular orientation are indicated in Fig. 1b. Analysis of the latter could lead to misinterpretation of the number of layers forming the outer capsid. Comparison of the clear edges of the virion in Fig. 1b and the high-power image (Fig. 1f) with mitochondrial membranes (Fig. 1a and e) reveals that the outer layer of the virion appears to be a single lipid bilayer coated by a thicker electron-dense layer. The electron-dense layer is the capsid and is composed mostly of the major capsid protein p73 (3).

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FIG. 1. Images of virus and mitochondria prepared using chemical fixation, taken at a magnification of x30,000. (a and c) Mitochondria consist of five domains: (i) outer membrane, (ii) intercristal space, (iii) inner membrane, (iv) cristae, and (v) matrix. (b and d) Whole ASFV which have been cut so that two sides appear parallel to the electron beam (arrows) and show a single membrane coated in viral protein; two appear nonparallel (arrowheads), in which case it is impossible to distinguish any layers. Each pair of images (a-b and c-d) were taken from the same infected cell. (e) Three-times digital zoom of an example of the inner and outer mitochondrial membranes, showing the "tramline" structure of each individual membrane. The membranes are not closely apposed. (f) An example of a viral envelope at three-times zoom. The virus appears to consist of a single membrane coated in capsid protein. The white bar indicates an example of the region where membrane measurements were taken. Scale bar, 10 nm.

Figure 1C and D compare a mitochondrion and virus from another cell after chemical fixation at a magnification of x30,000. Again the mitochondrial membranes appear as two dark lines (Fig. 1c), whereas the envelope of the ASFV virion appears as a tramline (Fig. 1d). We measured the thickness of viral envelope and outer mitochondrial membranes from a number of images from different cells (n = 20). The outer mitochondrial membrane measured 5.83 ± 0.59 nm whereas the viral envelope measured 6.34 ± 0.49 nm. If the viral envelope was composed of two membranes, then we would predict that its width should be roughly double that of a single mitochondrial membrane, i.e., 10.5 to 13 nm; however, this was not the case. This supports our suggestion that the intracellular viral envelope consists of a single bilipid layer.

Next we examined images of ASFV-infected cells that had been prepared from thawed cryosections (Fig. 2). Virus and mitochondria look very different from those prepared by chemical fixation (Fig. 1) because the stain used in cryosectioning produces a negative contrast (28). The effects of negative contrast are illustrated in the images of mitochondria (Fig. 2a, c, and e). The membranes appear light against the matrix background, while in chemically fixed mitochondria the membranes appear dark against the matrix (Fig. 1a, c, and e). The virions shown in Fig. 2b and d appear to have two similar-looking outer layers (Fig. 2b, d, and f). With this method of preparation, it is feasible to conclude that the virus is surrounded by a double membrane layer, as reported previously (1, 23). However, the outer layer visible in this preparation could also be the thick layer of capsid protein surrounding the viral envelope. The capsid protein does not have the same electron density as seen in positively stained material (Fig. 1b and d) because the negative stain does not interact directly with the protein but settles around it instead (14). Therefore, measurements of membrane thickness were taken excluding the dark edges, whereas membranes in positively stained material (with chemical fixation and high-pressure freezing) were measured including the dark edges. The width of the outer mitochondrial membranes in thawed cryosections (n = 20) was 4.70 ± 0.60 nm and the width of the viral envelopes was 5.18 ± 0.88 nm. As seen with chemically fixed cells, the measurements were consistent with the viral envelope being a single lipid bilayer.

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FIG. 2. Images of virus and mitochondria prepared using cryosectioning, taken at a magnification of x30,000. (a and c) The mitochondrial double membranes appear light, in negative contrast against the dark matrix. (b and d) Whole mature virions appear to be surrounded by two distinct layers of approximately equal size (arrows). The virions appear in negative contrast compared to those in Fig. 1 and 3. This is because of the staining method used in the preparation procedure. Mitochondria and virus in panels a to d are from the same section of prepared material. (e) Three-times digital zoom of an example of mitochondrial membranes. The two layers are indistinct but visible in negative contrast. (f) An example of a viral envelope at three-times zoom. The virus consists of two layers; however, the outer layer looks slightly larger than the inner layer. The white bar indicates an example of the region where membrane measurements were taken. Scale bar, 10 nm.

We recently developed a method for the rapid freeze-substitution of high-pressure-frozen cells which produces images with great detail and clarity when viewed in the TEM (13). Virus and mitochondria prepared using this method appear in positive contrast (Fig. 3), but note the increased definition of membrane structures compared to that seen after chemical fixation (Fig. 1). Each mitochondrial membrane appeared as a tramline at a magnification of x30,000 (Fig. 3a and c), and these are clearly visible when imaged at higher power (Fig. 3e). The membranes of the mitochondria appear to be better preserved than in either of the previous preparation methods, as there are fewer distortions and the membranes are more closely apposed (Fig. 3a; compare Fig. 1e and 3e), implying an absence of detrimental osmotic effects (20). Close inspection of the outer layers of whole virions (Fig. 3b and d) reveals that a single membrane is surrounded by a thick layer of capsid protein. High-power images clearly show a tramline coated with dense material, and again this is consistent with a single membrane surrounded by a protein layer (Fig. 3f). The width of outer mitochondrial membranes in high-pressure-frozen cells (n = 20) was 5.23 ± 0.37 nm, and the width of the viral envelope was 5.60 ± 0.66 nm. As was the case with chemically fixed and thawed cryosections, measurements of high-pressure-frozen membranes were consistent with the viral envelope being a single lipid bilayer.

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FIG. 3. Images of virus and mitochondria prepared using high-pressure freezing and freeze substitution, taken at a magnification of x30,000. (a and c) Mitochondria in this preparation have smoother membranes than those in Fig. 1a and c, and they are more closely apposed (arrowheads), indicating reduced extraction and osmotic artifacts. (b and d) Whole ASFV clearly consist of a single membrane (arrow) surrounded by a dense protein layer (arrowhead). This method of preparation preserves the samples in as near the natural state as possible. Each pair of images (a-b and c-d) were taken from the same infected cell. (e) Three-times digital zoom of an example of mitochondrial membranes. They are more closely apposed than in Fig. 1a and c but are still two distinct layers. (f) An example of a viral envelope at three-times zoom. The virus clearly has a single membrane coated with dense protein. The white bar indicates an example of the region where membrane measurements were taken. Scale bar, = 10 nm.

Measurements of viral and outer mitochondrial membranes after each of the three preparation methods were analyzed statistically, and the results are summarized in Table 1. Analysis of the data (by analysis of variance followed by Tukey's test) showed that there is a significant difference between the mitochondrial membrane measurements in each preparation, as well as between the chemical and cryosectioned or high-pressure-frozen virus membrane measurements (not shown). There is no significant difference between viral membrane measurements in cryosections or high-pressure-frozen material (not shown). This is unsurprising, as each preparation method will influence the subcellular and macromolecular structures of the cell to different extents. The mean values of membrane widths after each preparation show that the outer mitochondrial membrane is always smaller than the virus membrane. However, the ratio between virus envelope and outer mitochondrial membrane is roughly consistent throughout all three preparations (Table 1). This indicates that any difference between preparation techniques is due to differential shrinkage, while the structures remain constant.

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TABLE 1. Comparison of the widths of outer mitochondrial membranes and viral envelopes after different preparation methods

Statistical analysis of all the available data enabled us to reject the hypothesis that the virus envelope and cellular membrane (represented by the outer mitochondrial membrane) have different thicknesses (Table 1).

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The envelope of intracellular ASFV consists of a single bilipid layer. A thorough examination of the structure of intracellular ASFV by TEM demonstrated that the envelope is a single lipid bilayer. Infected cells were prepared by three different techniques: chemical fixation, cryosectioning, and high-pressure freezing. The clearest results came from high-pressure freezing, where membranes could be seen at a magnification of x30,000 (Fig. 3a and c), whereas after chemical fixation they became clear only at higher magnification (Fig. 1e). Infected cells prepared using high-pressure freezing were stabilized instantaneously, thus minimizing detrimental extraction, rearrangement, or osmotic effects. Of the three preparation methods used, this technique preserves the samples in as near the natural state as possible (13, 26). Close inspection of the viral envelope and capsid layer revealed a tramline surrounded by an electron-dense layer. We contend that the tramline represents a single lipid bilayer and that the electron-dense layer is the capsid composed of viral structural proteins. Interestingly, membranes prepared by cryosectioning appeared radically different from those prepared by the other two methods. The envelope and capsid layer in the cryosectioned material appeared as parallel electron-lucent structures suggesting, as recorded previously (23), a double membrane. The negative contrast produced by this preparation technique makes it difficult to differentiate between a membrane and a thick protein coat. Given that the viral envelope lies beneath the capsid protein p73 (2), the two layers visible in cryosectioned material probably represent the viral envelope and the coat of capsid protein.

Membrane measurements are consistent with the viral envelope being a single lipid bilayer. Calculations of the widths of the membranes of cellular organelles in conventionally fixed, epoxy resin-embedded material have been documented previously (16). The membranes of the nuclear envelope, ER, Golgi apparatus, and mitochondria and plasma membranes are all close to 5 nm. Our measurements of outer mitochondrial membranes are consistent with these observations. Measurements taken of the viral envelopes from all three preparation techniques showed that the envelope width varied from 5.18 ± 0.88 nm (for cryosectioning) to 6.34 ± 0.49 nm (for chemical fixation). The thickness of the viral envelope is therefore very similar to that of the single lipid bilayers of cellular organelles (16). The variation in width is not surprising, as all three techniques still involve some manipulation to get samples into the microscope. The exact size of the membrane is therefore less important than the ability to compare virus and mitochondrial membranes in the same image. The ratio of mean viral to outer mitochondrial membrane width in each preparation is roughly the same (1:0.9) (Table 1).

Assembly and envelopment of ASFV parallels the formation of intracellular VV IV. The results presented here suggest strongly that, in common with VV (15), the intracellular envelope of ASFV consists of a single lipid bilayer. The outer curvature of the envelope of VV IV is covered by a geodetic protein coat thought to contain the major capsid protein D13 (27). This has led to the suggestion that capsid assembly stabilizes the lipid bilayer before it closes into the sphere of the IV. Our early work has shown that the major capsid protein of ASFV is also recruited onto membranes, where it is incorporated into a large complex suggestive of a viral capsid (6, 7). One interpretation of these results is that the capsid protein of ASFV forms the protein coat seen above the envelope of the virus and that this also stabilizes the envelope during virus maturation. Unlike VV, which forms spherical and then brick-shaped particles, the capsid coat of ASFV is able to force the envelope into an icosahedron, which appears as an angular six-sided structure in cross section. As with VV, the origins of the ASFV envelope remain obscure. Heuser (15) suggested that single-membrane viral crescents of VV grow from T-shaped membrane junctions at a closed cellular compartment, for example, the ER. Such observations are consistent with the identification of ER- and ER-Golgi-intermediate compartment-derived membrane tubules and vesicles near VV crescents. Interestingly, recent work shows that if the VV A9 membrane protein is targeted to the ER, it will subsequently become incorporated into the inner envelope of the virus (17, 18). This suggests that even though a direct continuity of membranes between cellular compartments and the viral envelopes has been difficult to demonstrate (22), there is communication between the ER and the lipid envelope of VV. Similarly, one possible source of the ASFV envelope is the ER. However, incorporation of ER cisternae into virions during assembly would require the loss of one ER membrane by an as-yet-unidentified mechanism.

 
This work was supported by BBSRC.

 
* Corresponding author. Mailing address: Bioimaging, Pirbright Laboratory, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom. Phone: (44)1483 232441. Fax: (44)1483 232448. E-mail: pippa.hawes{at}bbsrc.ac.uk

Published ahead of print on 11 June 2008.

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Journal of Virology, August 2008, p. 7905-7912, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00194-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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