Electron Tomography of Nascent Herpes Simplex Virus Virions

Cells infected with herpes simplex virus type 1 (HSV-1) wereconventionally embedded or freeze substituted after high-pressurefreezing and stained with uranyl acetate. Electron tomogramsof capsids attached to or undergoing envelopment at the innernuclear membrane (INM), capsids within cytoplasmic vesiclesnear the nuclear membrane, and extracellular virions revealedthe following phenomena. (i) Nucleocapsids undergoing envelopmentat the INM, or B capsids abutting the INM, were connected tothickened patches of the INM by fibers 8 to 19 nm in lengthand $≤$ 5 nm in width. The fibers contacted both fivefold symmetricalvertices (pentons) and sixfold symmetrical faces (hexons) ofthe nucleocapsid, although relative to the respective frequenciesof these subunits in the capsid, fibers engaged pentons morefrequently than hexons. (ii) Fibers of similar dimensions bridgedthe virion envelope and surface of the nucleocapsid in perinuclearvirions. (iii) The tegument of perinuclear virions was considerablyless dense than that of extracellular virions; connecting fiberswere observed in the former case but not in the latter. (iv)The prominent external spikes emanating from the envelope ofextracellular virions were absent from perinuclear virions.(v) The virion envelope of perinuclear virions appeared denserand thicker than that of extracellular virions. (vi) Vesiclesnear, but apparently distinct from, the nuclear membrane insingle sections were derived from extensions of the perinuclearspace as seen in the electron tomograms. These observationssuggest very different mechanisms of tegumentation and envelopmentin extracellular compared with perinuclear virions and are consistentwith application of the final tegument to unenveloped nucleocapsidsin a compartment(s) distinct from the perinuclear space.

INTRODUCTION

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Introduction

Electron microscopic studies have shown that cells infectedwith herpesviruses contain four types of capsids. Procapsids,believed to represent the progenitor of other capsid types,contain a porous outer shell that is roughly spherical, surroundingan inner protein shell or scaffold (23, 33). Type A capsidsconsist mostly of a 125-nm-diameter protein shell that comprises12 vertices of fivefold symmetry (termed pentons) and 20 identicalplanar facets (53). Collectively, the planar facets are composedof 150 sixfold symmetrical subunits or hexons. Type B capsidshave an outer protein shell that is indistinguishable from thatof A capsids but also contain an inner, roughly spherical proteinshell (19, 52). Type C capsids, or nucleocapsids, also containan identical outer shell but lack the inner shell, which isreplaced with the densely packed linear double-stranded DNAgenome (5, 49).

In a process unique to herpesviruses, nucleocapsids bud throughthe inner nuclear membrane (INM) of infected cells into theperinuclear space, a compartment that is continuous with thelumen of the endoplasmic reticulum (ER), to become virions.The envelope of these nascent virions is therefore derived fromthe INM. In alphaherpesviruses, nucleocapsids are envelopedpreferentially over A or B capsids (40). In one model of virionegress, the virion envelope derived from the INM fuses withthe outer nuclear membrane (ONM), dumping the nucleocapsid intothe cytoplasm, where it subsequently undergoes a second envelopmentstep(s) in the Golgi apparatus, in the trans-Golgi apparatusnetwork, or in vesicles derived from these compartments (6,31, 45). Others have proposed that some nucleocapsids pass throughexpanded nuclear pores, receiving tegument prior to or at thetime of budding into other organelles such as the ER or Golgiapparatus (28, 48). (For purposes of discussion this will betermed the expanded nuclear pore model.) In both of these cases,the final virion envelope is (in the deenvelopment model) orcan be (in the expanded nuclear pore model) derived from organellesother than the INM. Alternatively, it has been proposed thatone or more enveloped virions bud from the ONM, forming transportvesicles. Fusion of the outer membrane of the transport vesiclewith the Golgi apparatus or other membranes would deliver envelopedvirions into the Golgi apparatus lumen for glycoprotein processingor, upon fusion with the plasma membrane, would deliver virionswithin the vesicle to the extracellular space (26). Thus, inthis model, the original virion envelope derived from the INMis retained throughout virion egress.

The space between the nucleocapsid surface and the inner surfaceof the virion envelope is termed the tegument. The tegumentof mature extracellular virions contains approximately 20 differentproteins of varying stoichiometry, and electron tomography hasshown that the tegument is arranged asymmetrically around thenucleocapsid as a series of concentric rings (22). Attachmentsof the innermost layer of mature tegument to capsid pentonshave been observed, suggesting that they serve to anchor thetegument layer to the nucleocapsid (51). The means of attachmentof the tegument to the virion envelope is uncertain, althoughinteractions between some tegument proteins and some glycoproteinshave been detected (8, 12, 17, 21).

The structure of the nucleocapsid has been determined to a resolutionof 0.85 nm from electron cryomicroscopic studies, and this structurepresumably reflects that of nucleocapsids within perinuclearvirions (52). Other features of perinuclear virions, however,are poorly characterized. Transmission electron microscopicstudies have indicated that perinuclear virions are morphologicallydistinct from extracellular particles. In particular, extracellularvirions contain prominent spikes protruding from the virionenvelope and a very densely stained tegument layer. Comparedto their counterparts in extracellular virions, the externalspikes of perinuclear virions are much less prominent, the tegumentappears much less dense, and the virion envelope often appearsdenser (20). It is logical to propose that the tegument of perinuclearvirions is relatively less dense because it contains less proteinthan its counterpart in extracellular virions, but this differencehas not been well defined. In any event, these observationssuggest that tegument proteins are applied to an unenvelopednucleocapsid, thus supporting either the expanded nuclear poreor deenvelopment model of virion egress.

Because perinuclear virions are difficult to separate from isolatedcytoplasmic and extracellular virions, few studies have beenundertaken to characterize the biochemical composition of perinuclearvirions. By immunogold analyses, it has been determined thatperinuclear virions and the INM of infected cells contain atleast the integral membrane glycoproteins B, C, D, and M (gB,gC, gD, and gM, respectively), a myristylated and palmitoylatedprotein encoded by U_L11, VP16 (at least when fused to greenfluorescent protein), and a protein complex containing the integralmembrane protein pU_L34 and its interaction partner pU_L31 (1,2, 9, 24, 24, 32, 39, 47). Interestingly, the U_L31 and U_L34proteins are largely absent from extracellular virions, suggestingthat the initial virion envelope derived from the INM is replacedduring virion egress (18, 39).

Under the hypothesis that such information would provide insightinto the assembly pathway of herpesviruses and the envelopmentreaction in particular, the major goal of this study was togenerate a three-dimensional structure of nascent virions undergoingenvelopment at the INM.

MATERIALS AND METHODS

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Materials and Methods

At 16.5 h postinfection, herpes simplex virus type 1 (HSV-1)[strain HSV-1(F)]-infected Vero cells were rinsed with serum-freeDulbecco modified Eagle medium and then scraped into cold serum-freeDulbecco modified Eagle medium plus 150 mM sucrose and pelletedat 250 x g for 5 min. As much supernatant as possible was removedwithout disturbing the pellet. From this point on, the cellswere kept on ice.

Conventional fixation. Cells were fixed with 4% paraformaldehyde (Electron MicroscopySciences) and 0.25% glutaraldehyde (Electron Microscopy Sciences)in 0.1 M NaP buffer, pH 7.4, for 30 minutes at room temperatureand then 90 minutes at 4°C. Cells were washed three timesfor 5 min each with 0.1 M sodium phosphate buffer and then dehydratedwith a graduated series of ethanol concentrations (10%, 30%,50%, 70%, and 100%) at 4°C and then –20°C. Thiswas followed by stepwise infiltration with Epon 812 resin (ElectronMicroscopy Sciences catalogue no. 14381) over the course of48 h at –20°C. Samples were aliquoted into gel capsules,and the resin was polymerized by exposure to UV light.

High-pressure freezing/freeze substitution. A cell slurry was made by resuspending pelleted infected cellsin a small amount of medium. Microdialysis tubing (200-µmdiameter; Spectrum Labs) was filled with the cell slurry bycapillary action and immediately submerged in 1-hexadecene.The tubing was cut with a scalpel to fit Staehelin freezinghats (Ted Pella). Each hat was filled with four to six piecesof tubing. The remaining empty space was filled with 1-hexadecene.Samples were frozen with liquid nitrogen under high pressureusing a Bal-Tec HPM010 machine. Samples were then freeze substitutedin 0.5% glutaraldehyde with 0.1% tannic acid in acetone at –90°Cfor 24 h. The acetone was replaced three times at –90°Cfor 30 min each time. Substitution was continued with 1% OsO₄made from crystal and 0.1% uranyl acetate in acetone at –90°Cfor 24 h. The temperature was allowed to rise to –20°Covernight. The acetone was replaced in three sequential stepswith Epon 812/araldite, first at –20°C, then at 4°C,and finally at room temperature. Each replacement step took1 h.

Samples were put into 50/50 uncatalyzed Epon-Araldite-acetoneat room temperature overnight and were then changed to 100%uncatalyzed Epon-Araldite for 24 h. Samples were then placedinto catalyzed Epon-Araldite for 8 h and then changed into freshcatalyzed Epon-Araldite and cured at 60°C for 48 h.

Electron tomography and three-dimensional reconstruction. Sections were cut at a 220-nm thickness and collected on Formvar-carbon-coated200-mesh copper grids. Grids were poststained with 2% uranylacetate for 20 min and Reynolds lead citrate for 5 min. Dual-axistomographic tilt series were carried out with an angular rangeof 120° and a tilt interval of 2°. Tilt images wererecorded with a JEOL JEM-4000FX operated at 400 kV in zero-lossmode using a Gatan GIF 2002 imaging energy filter. The imagepixel size relative to the specimen was 1 nm. The total electrondose for a dual-axis tilt series was about 10,000 e–/nm².

Tilt images were aligned, and tomographic reconstructions werecomputed using SPIDER (16, 36). The limits to resolution inthe reconstructions, set by data collection geometry and specimensize, were approximately 5 to 6 nm in the section plane and7 to 8 nm normal to the section (15). Tomograms were examinedslice by slice using WEB (34) and ImageJ (http://rsb.info.nih.gov/nih-image),and 1.0-nm-thick slices were selected for display. Surface-renderedmodels were made using Amira. The three-dimensional models showncame from a single representative tomogram.

RESULTS

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Results

HEp-2 cells were infected with HSV-1(F) at 5.0 PFU per celland at 16.5 h after infection were either Epon embedded andprocessed conventionally or high-pressure frozen and freezesubstituted. In both cases, three-dimensional (tomographic)images were reconstructed from tilt series of recorded images.

Although features in the tegument layer of high-pressure-frozenfreeze-substituted samples were similar to those observed insamples processed conventionally, the appearance of DNA withinthe nucleocapsid varied considerably in samples prepared usingthe different techniques. High-pressure-frozen, freeze-substitutedspecimens contained densely stained cores that mostly filledthe inner cavity of the nucleocapsid, whereas conventionallyfixed material contained considerably more space between theinner surface of the nucleocapsid and the electron opaque core(compare virion in Fig. 6 that was fixed conventionally withvirions in Fig. 1, 3, and 5, which were derived from high-pressure-frozen,freeze-substituted material). As a result of these observationsand the implication that freeze substitution was less proneto fixation artifacts, features in tomographic reconstructionsdetailing virion morphology were always verified in freeze-substitutedspecimens.

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FIG. 6. Electron tomographic reconstruction of virion in the perinuclear space and putative transport vesicle. Cells were infected with HSV-1(F) and were embedded and stained conventionally. The number of the slice in the reconstruction (of 150 total) is indicated in each image. The membrane of the putative transport vesicle is indicated with a large arrow. The position of the ONM is indicated with a small arrow.

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FIG. 1. One-nanometer-thick slices from a tomographic reconstruction of a type B capsid attached to the INM. Cells infected with HSV-1 were high-pressure frozen and freeze substituted. The slice number of each image (out of a total of 180 slices) is indicated in the lower left of each panel. The cytoplasm is at the top of the image, and the nucleus is at the bottom. An arrow indicates a single bridging rod emanating obliquely from the capsid surface. Cyto, cytoplasm; Nuc, nucleus.

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FIG. 3. One-nanometer-thick slices from a tomographic reconstruction of a nucleocapsid completing envelopment at the INM. The sample was prepared as for Fig. 1. The slice number of each image (out of a total of 235 slices) is indicated in the lower left of each panel. The position of the INM is indicated with an arrow. Triangles in slice 120 indicate two adjacent angular vertices with bridging rods, whereas the relatively planar surface between them is largely devoid of bridging rods. Nuc, nucleus; Cyto, cytoplasm.

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FIG. 5. One-nanometer-thick slices from a tomographic reconstruction of an extracellular virion. The sample was prepared as described in the legend to Fig. 1. The number of the section (of a total of 180) is indicated at the bottom of each image.

Initial studies focused on capsids abutting the INM. As notedby others viewing single thin sections, capsids both containingand lacking DNA were observed abutting patches of the INM (20,48). Nucleocapsids (capsids containing DNA) simply abuttingthe INM or that were partially enveloped at the INM were rare,but fully enveloped nucleocapsids within the perinuclear spacewere relatively common. B capsids lacking DNA and abutting theINM were also relatively common, but only occasionally werethese fully enveloped in the perinuclear space.

The patches of INM abutting B and C capsids were often markedlymore densely stained than the rest of the INM (Fig. 1), althoughin some preparations this was less apparent, presumably becauseof variability in staining from section to section. The patchesof INM abutting capsids were often indented. These indentationswere generally C shaped in single slices taken through an equatorialplane of the capsid (Fig. 1), and in three-dimensional reconstructions,they were generally cup-like, with the densely stained membraneextending over the full area that was adjacent to the capsid.(Fig. 1, and models Fig. 2C and 2D; see also movie S1 of rawdata in the supplemental material).

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FIG. 2. Models of the tomogram illustrated in Fig. 1. A. Model created by computationally fitting surfaces to densities in the tomogram, representing the capsid surface (purple) and the thickened region of the INM (green). Bridging rods (yellow) were segmented by manual tracing in sequential 1-nm sections. B. Similar to panel A, but the INM is made translucent to better illustrate the attachments of the bridging rods. C. Side view of a model created by manual tracing and surface rendering of the resulting contours. The inner and outer surfaces of the capsid (purple) and the INM (green) are shown, with the bridging rods (yellow) connecting the outer surface of the capsid with the inner surface of the thickened region of the INM. D. Same as panel A, but viewed from the cytoplasmic side to show the orientation of the bridging rods relative to the capsid surface. The inner protein sphere or scaffold does not appear in the model.

Capsids were not observed to directly contact the INM, or theycontacted the INM only rarely. Rather, the INM was usually separatedfrom the capsid surface by a consistent distance of approximately10 nm (Fig. 1). This distance was similar to that maintainedbetween the virion envelope and nucleocapsid of virions locatedbetween the INM and ONM (see below). Of interest was the observationthat in some sections, thin fibers could be visualized thatseemed to bridge the nucleocapsid surface and INM (Fig. 1; seealso movie S1 in the supplemental material).

In order to examine the bridging fibers relative to the virionsurface in more detail, the fibers in individual slices of agiven reconstruction (Fig. 2) and in some cases the capsid surface(Fig. 2A and B) were traced by hand. In both cases, a featurewas traced in each slice only if it was apparent in three consecutiveslices. The resultant modeled reconstructions revealed the presenceof densely stained, rod-like structures bridging the capsidsurface and INM. The rods generally projected radially awayfrom the nucleocapsid surface, although many emanated obliquelyfrom the capsid surface, rather than at right angles (an exampleis indicated by an arrow in Fig. 1). By using the known diameterof the capsid (125 nm), the bridging rods were calculated tobe approximately 8 to 19 nm in length with a thickness at orbelow the resolution of the reconstruction, about 5 nm in theplane of the sections. In most instances, the rod cross sectionswere elongated by about a factor of 1.3 in the normal direction,a result of the limited range over which the sections were tiltedin the electron microscope (±60°). The longest rodswere generally located at points within the interface of capsidand INM that were farthest from the center of the capsid. Thelength of the rods corresponded roughly to the width of thetegumental space located between the nucleocapsid surface andthe inner surface of the virion envelope as revealed in tomogramsof perinuclear virions (see below). In general, the rods werelocated only at the interface between the INM and the nucleocapsidsurface (i.e., they did not emanate from regions of the nucleocapsidnot directly opposed to the INM).

The next set of reconstructions was generated to determine whetherfeatures of capsids engaging the INM were reflected in perinuclearvirions. Type C capsids within the perinuclear space that weremostly or completely enveloped by the INM in single thick sectionswere studied by electron tomography. Angular vertices were onlyoccasionally observed in tomographic slices. The spacing ofvertices (approximately 60 nm apart) was consistent with thatexpected of capsid pentons. In single thick sections, the distancebetween the nucleocapsid surface and inner surface of the virionenvelope varied from as little as approximately 4 to 5 nm nearthe angular portions of the capsid surface to approximately13 nm opposite relatively flat faces (Fig. 3 and 4; see alsomovie S2 in the supplemental material).

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FIG. 4. Models of the tomogram shown in Fig. 3. A. Thick slice through the center of the virion. The nucleocapsid shell (purple) and virion envelope (green) are modeled by computationally fitting surfaces to densities in the tomogram, while the ONM (blue), INM (green), and bridging rods (yellow) are modeled by manual tracing. The DNA within the core is not shown. B. Side view of the nucleocapsid surface (purple) and the attached bridging rods (yellow). The horizontal elongation of the rods is an artifact due to the limited tilt range of the tomogram. C and D. Cutaway views of the enveloping nucleocapsid shown as a surface-rendered model after manual tracing. The nucleoplasm is on the bottom and cytoplasm on the top. The near portion of the model has been cut away for illustration. The nucleocapsid (purple), bridging rods (yellow), virion envelope (green), INM (green), and ONM (blue) are shown. As only the surfaces were rendered, the nucleocapsid and nascent virion envelope appear as hollow shells in this cutaway view. Note that the virion envelope is more than twice as thick as the neighboring INM.

Densely stained rods of approximately 2 to 5 nm in width and9 to 12 nm in length were seen bridging the outer surface ofthe nucleocapsid and the inner surface of the virion envelope.As described above, densities corresponding to individual rodsthat were discerned in at least three consecutive 1.0-nm sectionswere drawn by hand and included as yellow objects in the modelsshown in Fig. 4.

The rods were distributed asymmetrically on the surface of theotherwise roughly symmetrical nucleocapsid and projected atvarious angles from the nucleocapsid surface but generally radiallyaway from the nucleocapsid. Some rods projected obliquely fromthe nucleocapsid surface within the tegumental space. The rodslinked the virion envelope with both angular portions (Fig.3, indicated by triangles) and reasonably flat faces of thenucleocapsid. Many regions of the capsid surface also lackedobvious bridging rods, and these surfaces were more likely flattenedthan angular (Fig. 3, region between triangles). Distances betweensites of contact of the bridging rods varied between about 4and 13 nm along the nucleocapsid surface. With the caveat thatsome bridging rods may lie obliquely in the tegument and thereforemight contact sites other than their anchoring points in thenucleocapsid, the presence of bridging rod attachment pointsat distances closer than 64 nm (theoretical distance betweenpentons) argues that the bridging rods attach to faces of thecapsid (hexons) as well as their vertices (pentons). On theother hand, the observation that some planar surfaces were largelyspared of bridging rods argues that pentons are more frequentlyengaged than hexons.

Portions of the ONM bordering the nascent virion envelope protrudedinto the cytoplasm, with regions closest to the virion envelopeprotruding most dramatically (Fig. 3). Interestingly, at nopoint did the ONM contact the nascent virion envelope. Rather,the space between these membranes was maintained at a minimumdistance of approximately 15 nm.

Comparison of perinuclear and extracellular virions. Three-dimensional reconstructions of extracellular (Fig. 5)and perinuclear (Fig. 3 and 4) virions were compared, and thefollowing were noted. (i) The virion envelope in extracellularvirions was studded with rod-like densities that projected radiallyoutward from the virion envelope. These spikes or virion glycoproteinsvaried in shape and length but were up to about 20 nm long.They were generally narrower in regions closer to the virionenvelope and wider at more peripheral regions. Perinuclear virionsdid not have comparable projections. (ii) The tegument of extracellularvirions was much more densely stained than that of perinuclearvirions. The bridging rods of perinuclear virions that connectedthe nucleocapsid surface to the virion envelope were not readilyapparent in the tegument of extracellular virions. (iii) Thedistances and orientations of the nucleocapsid relative to thevirion envelope were asymmetric in both perinuclear and extracellularvirions. In the electron tomogram of the extracellular virionshown in Fig. 5, for example, some sections in the reconstructionsrevealed a spherical virion envelope whereas in others the virionenvelope was more pear shaped. In others (not shown) the virionenvelope contained a bulge of tegument at one pole, much aspreviously described (22).

Transport vesicles. Occasionally, single or multiple virions, surrounded by membrane,were noted in the cytoplasm near the nuclear membrane. Suchstructures have been proposed to represent transport vesiclesthat are separate from membranous organelles. These vesiclesare potentially significant because fusion of the vesicle withthe inner surface of the plasma membrane would be expected todeliver virions to the extracellular space, thus providing asimple model of virion egress. We therefore examined whetherstructures were discontinuous as seen in single sections orwere continuous with other organelles such as the ER or nuclearmembrane.

Three-dimensional reconstructions of thick sections revealedthat, of the putative transport vesicles located near the nuclearmembrane that were examined, all were in fact located withinextensions of the perinuclear space (Fig. 6). We were unableto substantiate the existence of distinct transport vesiclesnear the nuclear membrane in these studies and speculate thatmany of the structures noted previously represent cross sectionsof nuclear membrane with virions contained within the perinuclearspace.

DISCUSSION

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Discussion

Perhaps the most surprising finding in this study was the presenceof rods or fibers bridging the surface of the nucleocapsid andthe inner surface of the virion envelope. It should be notedthat at the present resolution the possibility that these bridgingdensities represent poorly resolved groups of two or more parallelrods is not precluded. It is tempting to speculate that theserods maintain the space between the virion envelope and nucleocapsidsurface, reminiscent of how a canvas tent (the virion envelope)is maintained by a series of poles (the bridging rods), embeddedin the ground (the nucleocapsid). The rods that bridge the innersurface of the virion envelope and nucleocapsid surface aremore prominent in perinuclear than extracellular virions, presumablybecause there is less obscuring stain density in the tegumentof the former. Alternatively, it is possible that the rods areunique to perinuclear virions derived from envelopment at theinner nuclear membrane and reflect a unique mechanism of virionbudding from the nucleus, as opposed to budding into cytoplasmicorganelles.

The identity of the protein(s) comprising the bridging rodsis not known, and it is not known if it corresponds to similar-appearingfibers noted in extracellular virions, speculated to be composedof filamentous actin (22). The viral proteins that are mostlikely to form the bridging rods would be components of perinuclearvirions that have a size sufficient to account for the observed8- to 19-nm length. The list of virally encoded proteins potentiallyfulfilling these criteria includes the pU_L31/pU_L34 protein complex.These proteins are particularly strong candidates because theU_L31 and U_L34 proteins are essential for envelopment at theinner nuclear membrane in a number of different cell lines andare known to become incorporated into perinuclear virions (7,29, 38, 41). U_L34 encodes a type II integral membrane proteinembedded in the INM with a predicted 27,265-Da portion of the29,789-Da protein located in the nucleoplasm, whereas the U_L31protein with an M_r of 33,951 directly interacts with U_L34 andanchors it to the nuclear lamina, possibly by also interactingwith lamin A/C (30, 37-39, 43, 44). Another possible candidateis the U_L25 protein that has been shown to associate with theexternal surfaces of purified capsids (27, 34, 46). Althoughample B capsids abut the INM in HSV-infected cells, C capsidsare preferentially enveloped (40). The presence of many capsidsabutting the INM of cells infected with a pseudorabies virusmutant lacking U_L25 argues against the possibility that U_L25is required to tether nucleocapsids to the INM (27). On theother hand, the observation that C capsids contain more U_L25than B capsids is consistent with the notion that U_L25 proteinmight be involved in selective envelopment of C capsids (42).

It is also possible that the bridging rods comprise one or moreproteins derived from the host cell rather than from the virus.A large number of cellular proteins potentially match the sizeand INM location of the rods observed in these studies includingactin, lamins, and lamin receptors. Actin is a strong candidategiven the presence of actin in herpesvirions, the associationof F actin with capsids in infected-cell nuclei, and the dependenceof active intranuclear movement of capsids on actin (3, 10,11, 13, 14, 25, 50). Lamins are less likely because they arelocally depolymerized to allow access of nucleocapsids to theINM; thus, lamin filaments would not be expected to become incorporatedinto nascent virions (4, 35, 37, 44). Efforts to identify thecomponents of the bridging rods are under way.

Previously noted differences between perinuclear and extracellularvirions fixed in glutaraldehyde and embedded in glycid ether100 (17) were confirmed in this work using high-pressure freeze-substitutiontechniques. Specifically, the tegument of perinuclear virionsis much less dense overall and the virion envelope is considerablydenser. Moreover, projections emanating from the external surfaceof the extracellular virion envelope are much more prominent,wider in diameter, and longer. Although the precise reasonsfor these differences are not known, it is logical to presumethat the extensive changes in the external spikes are a consequenceof more extensive glycosylation in the mature glycoproteinsof extracellular virions. The increased stain density of thetegument of extracellular virions also suggests that additionaltegument proteins are applied after the initial envelopmentstep at the INM. These data are therefore most consistent witha model in which the virion envelope initially obtained fromthe INM is lost in a subsequent fusion event (deenvelopment)and then replaced by budding into a cytoplasmic membrane compartment(reenvelopment), as originally proposed by others (45). Thesedata are also theoretically consistent with the possibilitythat the tegument is applied to nucleocapsids that pass throughexpanded nuclear pore complexes into the cytoplasm (28). Onthe other hand, we were unable to substantiate the expansionof nuclear pores as reported in previous studies, although oursearch was not exhaustive (28, 48).

One argument supporting the possibility that the original virionenvelope is maintained throughout virion egress has been thelongstanding observation that perinuclear virions can be observedwithin cytoplasmic vesicles. A simple model of virion egresssuggests that fusion of this transport vesicle with the plasmamembrane should deliver the enclosed virion to the extracellularspace. In this report, however, we noted that a number of apparentvesicles containing virions were, when followed through a three-dimensionalreconstruction, actually irregular extensions of the nuclearmembrane, with lumens continuous with the perinuclear spaceor endoplasmic reticulum. These observations are also consistentwith the deenvelopment model of virion egress. On the otherhand, we cannot exclude the possibility that distinct vesiclescontaining nascent virions exist; instead, we would argue thatif such transport vesicles do exist, they are more rare thanpreviously supposed. Electron tomograms of the entire nuclearmembrane and contiguous ER of infected cells would be usefulto confirm the existence of such vesicles.

ACKNOWLEDGMENTS

We thank V. Mandayam Parthasarathy and the staff of the CornellIntegrated Microscopy Center for useful discussions and helpwith the freeze substitution.

The studies at Cornell University were supported by NIH grantAI 52341 to J.D.B. The microscopy and image processing workwas supported by NIH grant RR01219 (J. Frank, Principal Investigator)through the NCRR Biomedical Research Technology Program.

FOOTNOTES

* Corresponding author. Mailing address: C5132 Veterinary Medical Center, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 253-3391. Fax: (607) 253-3384. E-mail: jdb11{at}cornell.edu.

Published ahead of print on 10 January 2007.

Supplemental material for this article may be found at http://jvi.asm.org/.

REFERENCES

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