Vol. 65, No. 3
JOURNAL OF VIROLOGY, Mar. 1991, p. 1066-1081 0022-538X/91/031066-16$02.00/0 Copyright C) 1991, American Society for Microbiology
Effect of Brefeldin A on Alphaherpesvirus Membrane Protein Glycosylation and Virus Egress M. E. WHEALY, J. P. CARD, R. P. MEADE, A. K. ROBBINS, AND L. W. ENQUIST* E. I. duPont de Nemours, Central Research and Development, Viral Diseases Group, Experimental Station, Wilmington, Delaware 19880-0328 Received 23 August 1990/Accepted 12 November 1990
In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting doublemembraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.
Pseudorabies virus (PRV;
a
It is not clear whether these two models are mutually exclusive or are indicative of multiple pathways for envelopment among alphaherpesviruses. To gain insight into this problem, we have examined the pathway for assembly and egress of PRV during the normal course of infection and following treatment with brefeldin A (BFA), an inhibitor of transport between the ER and the Golgi apparatus which ultimately leads to dissolution of the Golgi cisternae. BFA is a potent antiviral agent with marked effects on herpesvirus replication (35). It is now established that BFA disrupts the movement of newly synthesized membrane proteins into the Golgi apparatus (14) and that this effect is reversible (15). After treatment of the cell with BFA, Golgi enzymes are redistributed to the ER, where they remain active (15). Moreover, the Golgi apparatus of BFA-treated cells rapidly disappears as a morphologically distinct entity (14). We reasoned that if the Johnson and Spear model was correct for PRV, then a specific block in ER-to-Golgi apparatus transport should result in the accumulation of enveloped virions in the ER. Furthermore, if the Jones-Grose model was correct for PRV, then an inhibitor that destroys the Golgi apparatus should completely block the formation of intracellular enveloped particles. In this report, we show that BFA has dramatic effects on PRV membrane protein glycosylation, processing, and virus egress consistent with a pathway involving elements of both the HSV and VZV models. Our results are consistent with a multistep pathway for envelopment of PRV that initially involves acquisition of a membrane by budding of capsids into the perinuclear space followed by transport through the ER and deenvelopment, with release of the capsids into the cytoplasm in proximity to the trans-Golgi. The next step involves a second envelopment of these capsids by membrane derived from the transGolgi containing mature, fully processed viral glycoproteins. The resulting double-membraned virus is transported to the
swine pathogen), varicella-
zoster virus (VZV; a human pathogen), and herpes simplex virus type 1 (HSV-1; a human pathogen) are typical mem-
bers of the Alphaherpesvirinae subfamily of herpesviruses (28, 29). The envelope of VZV contains at least five glycoproteins (7), and the envelopes of PRV and HSV-1 contain at least seven virus-encoded glycoproteins (29, 33, 40). The function of the multiple viral glycoproteins, their targeting and proper assembly into the virus envelope, the site of initial virus envelopment, and the pathway of virus egress from infected cells present interesting and as yet only partially understood problems in herpesvirus biology. In general, herpesviruses are unique among enveloped viruses in that the primary site of envelopment of herpes virions is thought to be the inner nuclear membrane (1, 5, 6, 9, 16, 18, 19-21, 24, 33, 34). Johnson and Spear (9) suggested a model for HSV-1 envelopment and viral egress by which HSV-1 virions first were enveloped at the inner nuclear membrane, where they acquired viral glycoproteins lacking Golgi modifications. Subsequently, these "immature" virions were transported to the Golgi apparatus, where precursor glycoproteins were modified as the enveloped particle moved through the Golgi stacks. A contrasting model has been proposed for VZV by Jones and Grose (10), who suggested that VZV capsids were released from the nucleus into the cytoplasm by an unspecified mechanism and subsequently budded into post-Golgi transport vesicles, acquiring a "mature" envelope with fully modified glycoproteins. In this model, the mechanism by which VZV capsids leave the nucleus is unspecified, but virions do not enter the endoplasmic reticulum (ER) nor do they enter the Golgi stacks.
*
Corresponding author. 1066
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plasma membrane, where fusion releases a mature, enveloped virus particle from the cell.
MATERIALS AND METHODS Materials, cells and viruses. BFA was obtained from Epicentre Technologies, Madison, Wis. It was made up as a 1-mg/ml solution in ethanol and stored at -20°C. The Becker strain of PRV was prepared and used as previously described (26). The cell line used for PRV virus infections was PK15 cells (swine kidney fibroblasts). Growth curves. Single-step growth curves of PRV were done as described by Whealy et al. (38), with modifications as noted in the legend to Fig. 4. Antibody reagents. The antisera used in these studies included a mouse monoclonal antibody, 6D8MB4, reactive with gpSO (a kind gift from C. Whetstone, Ames, Iowa), a goat polyvalent antiserum (no. 282) raised against a denatured Escherichia coli-produced Cro-gIll fusion protein which is reactive with native and denatured glll protein (27), and a goat polyvalent antiserum (no. 284) which recognizes the native and denatured forms of the gII glycoprotein (25). Pulse-chase analysis. The pulse-chase procedure used has been described previously (30). Briefly, PK15 cells were infected at a multiplicity of infection of 10 with PRV Becker. Where indicated, 2.5 ,ug of BFA per ml was added at S h postinfection and was maintained throughout the experiment. The cells were deprived of cysteine at 5.5 h postinfection, and a 2-min radioactive pulse of 100 ,uCi of [35S]cysteine per ml was administered at 6 h postinfection. Radiolabel was then removed, and the cells were incubated in medium containing excess nonradioactive cysteine for various times as indicated in the figure legends. At the desired chase times, monolayers were harvested and samples were immunoprecipitated with specific antiserum. After immunoprecipitation, some samples were digested with the enzyme endoglycosidase H according to the specifications provided by New England Nuclear Corp. All immunoprecipitates were loaded onto sodium dodecyl sulfate (SDS)10% polyacrylamide slab gels. Fluorography was conducted with sodium salicylate (4) and was followed by autoradiography. Measurement of gIl oligomer formation by sedimentation through sucrose gradients. gIl oligomer formation was measured as described by Whealy et al. (39) with modifications as noted in the legend to Fig. 3. Electron microscopy. (i) Sample preparation. Paired samples of BFA-treated and untreated control cells were collected 5.5, 6, 7, and 8 h following infection of cultures with PRV. The protocol for infection of PK15 fibroblast cultures and treatment of cells with BFA reproduced that described for the pulse-chase analysis. At each time point, samples were immersed in a buffered aldehyde solution (5% transmission electron microscopy-grade glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium phosphate buffer [pH 7.4]) and fixed at room temperature for 1 h. Thereafter, samples were washed extensively with buffer prior to and following a 1-h postfixation in buffered 2% osmium tetroxide solution containing 1.5% potassium ferricyanide (13), dehydrated in a graded series of ethanol, and embedded in Epon plastic resin using standard methods. Thin (800-nm) plastic sections were cut perpendicular to the plane of the cell monolayer with a Reichert Ultracut E ultramicrotome. A series of sections was collected on Formvar-coated slot grids, stained with heavy-metal salts (uranyl acetate and lead citrate), and
1067
analyzed with either Hitachi 600 or JEOL 1200ex transmission electron microscopes. (ii) Analysis. Cells from each sample were systematically photographed and examined for the effects of BFA treatment at each time point. As noted above, paired samples of treated and untreated cells were included for each time point and processed in parallel to reduce variations in cellular preservation that might occur in the course of tissue preparation. Analysis of the tissue was conducted in three phases. First, 10 complete cells from each sample were photographed at an intermediate magnification of x 10,000, and photomontages of the cells were assembled at a final magnification of 25,000 diameters. Cells chosen for this analysis exhibited large cross-sectional areas of both the cell nucleus and the cytoplasm. Second, 430 higher-magnification (x 20 to x40,000) micrographs (approximately 50 per sample) were taken of representative areas of cells in each sample. We selected these areas to include long stretches of nuclear envelope and/or organelle-rich regions of cytoplasm. These negatives were enlarged 2.5 times for a final print magnification ranging from xS0 to x100,000. Third, having completed the first two phases of the analysis, we conducted a directed examination in which we photographed those features which were most characteristic of each time point and treatment group. Two aspects of the material were subjected to quantitative analysis, and comparisons were made between experimental and control groups. These included (i) the number of virions per unit membrane of nuclear envelope or ER (rough and smooth) and (ii) the number of viral particles (capsids and enveloped virions) in either the nucleus or the cytoplasm. The first analysis was restricted to linear (longitudinal) stretches of membrane measured with a cartographer's tool. RESULTS BFA does not affect PRV glycoprotein synthesis but blocks processing and export. Previous studies documenting the effects of BFA on cell metabolism and morphology were conducted in the absence of a viral infection. Consequently, we felt it was important to establish basic parameters of the effect of BFA on PRV membrane protein synthesis. This is necessary because PRV, like all alphaherpesviruses, significantly alters host cell metabolism after infection (2). We analyzed the synthesis and export of three well-characterized PRV membrane proteins by pulse-chase analysis. These three glycoproteins, gIl, gIll, and gp5O, were chosen because of specific posttranslational modifications that enabled us to judge the effects of BFA. Briefly, the gIl glycoprotein of PRV contains six N-linked glycosylation sites, forms oligomers in the ER, and, following transport to the Golgi, is processed by a protease (39). The gIll glycoprotein of PRV contains 8 N-linked glycosylation sites that are converted to complex forms in the Golgi, and gIll is likely to be modified by 0-linked glycosylation (30). Finally, the gpSO glycoprotein of PRV has been shown to contain only 0-linked and not N-linked oligosaccharide modifications (22). Infected cells were labeled for 2 min with [35S]cysteine (pulse), and then the isotope was removed and replaced with medium containing an excess of nonradioactive cysteine (chase). Samples were taken at various times and immunoprecipitated with PRV glycoprotein-specific antisera. Identical experiments were done in the presence of BFA. Infected cells were treated with 2.5 ,ug of BFA per ml for 1 h prior to the 2-min pulse. BFA was maintained at 2.5 ,ug/ml during the chase. The results of these experiments are shown
WHEALY ET AL.
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number of gll-related polypeptides. At the 15-min chase a 100-kDa species, representing the completed gIl molecule with high-mannose glycosylation, was apparent. This species was subsequently converted to a 110-kDa species after 45 min of chase. The 110-kDa species began to be processed to the 68- and 55-kDa species by about 45 min after the pulse. In the presence of BFA, the 2-min pulse profile remained essentially identical to that seen in the absence of inhibitor. Therefore, BFA did not affect initial synthesis and glycosylation of gll. However, conversion of the 100-kDa species to the 110-kDa species as well as subsequent protease processing of the 110-kDa species was completely blocked by BFA. The labeling kinetics of gpSO are shown in Fig. 1C. In the absence of BFA, the profile is essentially that reported by Petrovskis et al. (22). We noted that the gp5O monoclonal antibody did not recognize anything synthesized in the pulse, a result obtained with all the gpSO monoclonal antibodies we tested. A similar observation was reported by Petrovskis et al. (22). At 15 min after the pulse, a 46-kDa species was immunoprecipitated, and this form was rapidly converted to a 55-kDa species by 30 min after the pulse. A different profile was observed in the presence of BFA. At 15 min of chase, the 46-kDa protein was replaced by a novel 49-kDa species that was never converted to the 55-kDa form. Novel endoglycosidase H-resistant glll species arise after prolonged treatment with BFA. While BFA completely blocked the formation of predicted Golgi-modified forms of all three PRV glycoproteins, it is obvious that in the presence of BFA, the primary glycosylated species of glIl and gp5O begin to acquire new modifications that retard electrophoretic mobility. These species are never seen in untreated infected cells. This phenomenon is not unexpected. It has been established that BFA treatment of uninfected cells results in rapid redistribution of some Golgi proteins into the ER and that some of the Golgi enzymes retain activity in their new environment (13, 15). We considered the possibility that these forms result from the action of Golgi enzymes redistributed to the ER after BFA-induced dissolution of the Golgi apparatus. One of the Golgi modifications that may be occurring is the conversion of the high-mannose N-linked oligosaccharides to complex forms. This can be tested by measuring the endoglycosidase H sensitivity of the glycoproteins. We concentrated on glll for this analysis. The results are shown in Fig. 2. An aliquot from each of the samples used for Fig. 1A (glll) was digested with endoglycosidase H as described in Materials and Methods. Some aliquots were untreated and some were treated with BFA as described in the legend to Fig. 1A. Ryan et al. (30) demonstrated that the 74-kDa form of gIll was sensitive to endoglycosidase H, resulting in formation of a 58-kDa form, but the 92-kDa species was resistant to digestion. The results in Fig. 2 are essentially identical to these findings. In the presence of BFA (Fig. 2), the 74-kDa precursor was initially sensitive to endoglycosidase H digestion, giving rise to the expected 58-kDa deglycosylated form of gIII. However, as the chase continued in the presence of BFA, modifications appeared that could not be removed by endoglycosidase H. Enzyme digestion reduced the apparent molecular mass, but the final digestion product was larger than the expected 58-kDa deglycosylated form. In other experiments (data not shown), similar samples were also treated with endoglycosidase F, which should cleave both the high-mannose and the complex carbohydrates added to the N-linked sites. This enzyme reduced the apparent molecular mass more than endoglycosidase H treatment but did
point,
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FIG. 1. Pulse-chase analysis of PRV glycoproteins in the absence and presence of BFA. PK15 cells were infected with PRV at a multiplicity of infection of 10. At 5 h postinfection, BFA was added at a concentration of 2.5 ,ug/ml. The cells were pulse-labeled at 6 h postinfection and chased for the times indicated across the top in minutes. The cells were lysed and immunoprecipitated with gIllspecific serum (A), gll-specific serum (B), or gpSO-specific serum (C). Immunoprecipitates were resolved on a 10% SDS-polyacrylamide gel and visualized by fluorography. The molecular mass standards (in kilodaltons) are indicated to the left of each panel.
in Fig. 1. The left side of each panel contains the untreated controls, and the right side contains BFA-treated cells. Immunoprecipitations of glll, gll, and gpSO are shown. The primary product of glll synthesis was a 74-kDa protein fully glycosylated with high-mannose oligosaccharides (30). Figure 1A demonstrates that by 30 min of chase, the 74-kDa form was converted to a diffusely migrating species with an average apparent molecular mass of 92 kDa known to be resistant to endoglycosidase H and sensitive to endoglycosidase F (30). In the presence of BFA, the 74-kDa form was synthesized, indicating that initial high-mannose glycosylation was unaffected. However, the conversion of the 74-kDa form to the 92-kDa species was completely blocked. At late chase times in the presence of BFA, the 74-kDa species exhibited a slight but reproducible retardation in electrophoretic mobility. The labeling kinetics of glycoprotein gll are shown in Fig. 1B. In the absence of BFA, the profile is that described by Whealy et al. (39). The primary products of synthesis were a
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FIG. 2. Endoglycosidase H treatment of PRV glll. PK15 cells infected with PRV at a multiplicity of infection of 10. BFA (2.5 ,ug/ml) was added at 5 h postinfection where indicated. At 6 h postinfection, the cells were pulse-labeled and chased for the times indicated (in minutes). The cells were lysed and immunoprecipitated with gIll-specific serum. The immunoprecipitates were treated overnight with endoglycosidase H as described by the manufacturer prior to fractionation on a 10% SDS-polyacrylamide gel and visualization by fluorography. The molecular mass standards (in kilodaltons) are indicated to the left. were
not reduce it to that of the expected 58-kDa deglycosylated form. This indicates that there is some conversion of the high-mannose N-linked oligosaccharides to complex forms, presumably by Golgi enzymes that have recycled back to the ER after BFA treatment. In addition, other modifications, such as 0-linked sugars, may be added in the presence of BFA. Formation of primary gIl oligomers is not blocked by BFA treatment. Whealy et al. (39) demonstrated that gIl is initially synthesized in the ER as a monomer with high-mannose modifications. This 100-kDa precursor is converted to an oligomer (most likely a homodimer) by about 30 min. Subsequently, the precursor oligomer is converted to an oligomer of identical sedimentation value but composed of 110-kDa monomers by the partial conversion of the highmannose sugars to complex forms in the Golgi apparatus. Next, the mature oligomer is cleaved by a cellular endoprotease, resulting in a complex with the subunits held together both by disulfide and SDS-resistant interactions (39). On the basis of kinetics of export and processing (39), the cellular protease was deduced to be a late Golgi enzyme. In the first section, we determined that BFA blocked the conversion of the 100-kDa precursor to the 110-kDa species and that no protease processing of the precursor occurred. It was of interest to determine whether the 100-kDa monomers had been converted to oligomers. To determine this, infected cells were treated with 2.5 ,ug of BFA per ml at 5 h postinfection. At 6 h postinfection, cells were labeled for 2 min with [35S]cysteine and then chased for 10 and 60 min with excess cold cysteine as described by Whealy et al. (39). The 10- and 60-min chase samples were solubilized in Triton X-100 and fractionated on sucrose gradients as described by Whealy et al. (39). Gradient fractions were immunoprecipitated with anti-gIl serum, and the precipitates were analyzed on an SDS-polyacrylamide gel (Fig. 3). It is clear that in the presence of BFA, the gII highmannose-modified precursor of 100 kDa sedimented as a monomer at the 10-min chase point and by 60 min after synthesis was converted into oligomers cosedimenting with normal gII oligomers. These data support the suggestion of Whealy et al. (39) that primary oligomerization of gII occurs in the ER prior to export to the Golgi apparatus.
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BFA blocks formation of infectious particles as determined by single-step growth experiments. It was clear from the experiments described above that BFA had a dramatic effect on PRV glycoprotein maturation and export. As reported by others for uninfected cells, BFA caused a specific and essentially complete block of ER-to-Golgi apparatus transport as measured by lack of complex N-linked-sugar formation, completion of 0-linked-sugar modifications, and proteolytic processing of viral glycoprotein oligomers. Therefore it was of interest to determine the effects of BFA on virus particle formation. We first examined the effects of BFA on formation of infectious particles. For this experiment, a series of plates was infected at a multiplicity of infection of 5 and incubated at 37°C. At 1, 3, 5, 7, 9, 11, 13, and 24 h postinfection, infected cells and media were harvested and virus titers of the cell-associated and medium fractions were determined. At 5 h postinfection, 2.5 ,ug of BFA per ml was added to one set of plates. The results of this experiment are shown in Fig. 4A. In the absence of BFA, results were essentially identical to those described previously for this strain of PRV (38). However, upon the addition of BFA, production of infectious virus stopped abruptly, with the number of infectious particles remaining relatively constant for the duration of the experiment. The infectious virus remaining after BFA treatment must represent extracellular particles formed prior to the addition of BFA which adhere to the extracellular matrix since they are sensitive to low-pH citrate treatment (data not shown). This treatment is known to kill PRV particles outside cells but has no effect on virions inside the cell (17). A second experiment was done to determine whether the effects of BFA on infectious-particle formation were reversible. A single-step growth experiment was done as described above except that after 30 min of BFA treatment at 5 h postinfection, the drug was removed and replaced with drug-free medium for the duration of the experiment. Infected cells were scraped into the medium at 30-min intervals after the addition of BFA, and titers were determined for plaque-forming particles (Fig. 4B). As shown in Fig. 4A, the addition of BFA blocked the accumulation of infectious particles. After the removal of BFA, a lag of about 1 h ensued and then infectious particles began to accumulate at a rate similar to that of the untreated control. By 9 h postinfection, both treated and untreated cultures accumulated similar titers of infectious virus. We conclude that the effects of BFA on infectious-particle formation are reversible. Morphological consequences of PRV infection and effects of BFA treatment. BFA clearly stopped the formation of infectious virus, but at what stage were virus assembly and egress blocked? We used transmission electron microscopy to give us insight into this problem. To appreciate more fully the effects of BFA on PRV-infected cells, it was important to establish basic morphological parameters in infected but untreated cells. In what follows, representative electron micrographs will be referred to, but the conclusions are never drawn from a single micrograph. Specifically, at least 10 complete cells from each time point (5.5, 6, 7, and 8 h after infection) were examined, and each examination involved more than 50 higher-magnification micrographs of representative areas of the cell. The details of our analysis are described in Materials and Methods. Figure 5 illustrates the major morphological alterations that occurred in PK15 cells in response to PRV infection. Shown is a typical infected cell at 8 h after infection. Pathological invaginations of the nuclear envelope, disas-
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FIG. 3. Gradient fractionation of PRV gll. PK15 cells were infected at a multiplicity of infection of 10. BFA was added at a concentration of 2.5 ,ug/ml at 5 h postinfection. At 6 h postinfection, the cells were pulse-labeled and chased for 10 or 60 min as indicated. The cells were solubilized in 1% Triton X-100 and fractionated by sedimentation through a 5 to 15% sucrose gradient containing 0.1% Triton X-100. The fractions were immunoprecipitated with a gll-specific antiserum. The immunoprecipitates were resolved on a 10% SDS-polyacrylamide gel and visualized by fluorography. The positions of the molecular mass standards (in kilodaltons) resolved on the polyacrylamide gel are indicated to the left of each panel. The relative positions of the expected monomers and dimers of gll are indicated at the top.
sembly of the nucleolus, and clumping of chromatin were the hallmarks of infection. In addition, infected cells characteristically exhibited pronounced accumulations of capsids both in the nucleus and the cytoplasm (Fig. 5). Moreover, capsids in various stages of envelopment were commonly associated with the forming face of the Golgi apparatus. These morphological indicators increased dramatically as infection proceeded. For example, most cells at 5.5 h postinfection exhibited only a few fingerlike invaginations in the nuclear envelope, minimal clumping of the nuclear chromatin, and a moderate number of nucleocapsids dispersed throughout the nucleoplasm. However, by 8 h postinfection, multiple irregular nuclear invaginations analogous to those illustrated in Fig. 5 characterized the majority of infected cells, and large crystalline arrays of nucleocapsids typically were present adjacent to the nuclear envelope. It is important to note that only in rare instances did we observe virions between the inner and outer leaflets of the nuclear envelope. The cytoplasm contained a variety of morphologically distinct virus particles that accumulated as the infection proceeded. We noted that these particles exhibited a preferential association with the Golgi cisternae. Although individual virions occurred throughout the cytoplasm, the majority of particles were concentrated in focal aggregations immediately adjacent to the trans cisternae of the Golgi apparatus (Fig. 6). These areas characteristically consisted of a com-
plex array of capsids, immature virions, and bilaminar membrane profiles. Rough ER membranes were prevalent in the vicinity of these areas and in some instances were found in the midst of the virion aggregations (Fig. 6, 7a, and 8). However, most membranous profiles had no associated ribosomes and in many instances were continuous with the trans-cisternae of the Golgi apparatus (Fig. 6 and 7a). These membranous arrays surrounded individual capsids and ultimately fused to form a bilaminar envelope (Fig. 6 and 7). The resulting "virions" were composed of central capsids surrounded by two distinct membranes, with an average crosssectional diameter of approximately 200 nm. To confirm that capsids were principally associated with the trans-cisternae of the Golgi apparatus, we examined serial thin sections through the regions of virion aggregation (Fig. 8). This analysis demonstrated that several Golgi complexes were associated with an individual aggregation. This is clearly evident in the consecutive sections illustrated in Fig. 8: seven different Golgi complexes are intimately associated with a single prominent region of capsid aggregation immediately adjacent to the cell nucleus. With the exception of one complex (no. 2), all of the Golgi bodies were found at the periphery of the capsid aggregation. In addition, the trans-cisternae of all of the profiles are oriented toward the virion aggregation, and there are a number of clear examples of extensions of the trans-cisternae sur-
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FIG. 4. Single-step growth curves of PRV. PK] infected with PRV at a multiplicity of infection of 5 an d incubated at 370C. (A) At 1, 3, 5, 7, 9, 11, 13, and 24 h postinfectio n, plates were harvested and the virus titers of the cell (0) and me dium fractions (Q) were determined separately. At S h postinfecti ion, BFA (2.5 mg/ml) was added to a duplicate set of plates (-) ancJ harvested as described in the text for untreated cells. Data for BFA-treated cell-associated samples (-) and medium samples (LI) a3re shown. (B) At 1 and 3 h postinfection, the infected cells were sciraped into the medium and titers were determined. At 5 h postinfecti ion, half of the plates were treated with BFA at a concentration of 2.5 j±gIml (T).At 5.5 h postinfection, the medium was removed and disc arded from all plates, the monolayers were washed with phosphate-b uffered saline, and fresh Dulbecco modified Eagle medium was ad led (O). Cells were harvested at 5, 5.5, 6, 6.5, 7, 7.5, and 9 h po stinfection as described in the text for 1 and 3 h (&, no BFA; A 30-min BFA treatment). The scale on the left is the log of the p laque-forming units per milliliter.
rounding individual capsids (see Golgi complex no. 3 in Fig. 8a and b). BFA treatment resulted in rapid dissolution of the Golgi cisternae and a dramatic accumulation of viri(cns between the inner and outer nuclear membranes as well aas in the ER. Other than loss of the Golgi structures and acciumulation of enveloped virions, no alterations in cellular morphology were visible. These effects were evident withiin 30 min of application of the drug and became more dramattic with time. In all instances, prominent accumulations of virions were apparent between the internal and external le aflets of the nuclear envelope (Fig. 9a and b and 10a thr ough c) and within the rough ER of the cytoplasm (Fig. 9c). Quantitation of the number of virions in the nuclear envelc pes and the
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rough ERs of control and experimental samples at each time point provided a striking illustration of this effect (Fig. 11). At 30 and 60 min following BFA treatment, we saw no difference in the number of virions per 10 ,um of membrane in either the nuclear envelope (Fig. 11A) or the ER (Fig. 11B). However, at 2 and 3 h after BFA treatment, we observed a marked increase in the number of virions in each of these compartments that was not apparent in paired control samples. The number of virions in the nuclear envelope increased from approximately 0.5 virions per 10 ,um of membrane at 30 min post-BFA treatment to more than 7 virions per 10 ,um at 3 h posttreatment (Fig. 11A). A similar but less dramatic increase occurred in the ER (Fig. 11B). Since the linear extent of ER far exceeds that of the nuclear membrane, this is a predicted result. These differences occurred even though the relative number of capsids and enveloped virions per cell remained relatively constant in both untreated and BFA-treated infected cells (data not shown). The morphology of virions apparent between the inner and outer nuclear membranes of BFA-treated cells varied considerably. We observed structures containing profiles of relatively immature capsids surrounded by a single membrane to capsids surrounded by a prominent tegument and a dense envelope reminiscent of that observed for mature virus in untreated cells. Numerous micrographs showed that the less-mature form of virion acquired its membrane by budding through the inner nuclear membrane (Fig. 10a and b). Similarly, we observed several examples of capsids gaining access to the cytoplasm by fusion of this membrane with the outer leaf of the nuclear envelope (Fig. 10c). This observation was consistent with the presence of numerous free capsids within the cytoplasm of BFA-treated samples. However, the location and organization of these free cytoplasmic capsids differed from those observed in paired controls. In essentially every case, most capsids observed in the cytoplasm of BFA-treated cells were concentrated around spherical accumulations of dense granular material (Fig. 10d). These granular matrices were more commonly observed in BFA-treated cells and appeared to arise coincident with the dissolution of the Golgi cisternae. DSUSO DISCUSSION
BFA blocks PRV glycoprotein processing and export. BFA dramatically effected PRV membrane glycoprotein processing and export. We examined three unique viral membrane glycoproteins that undergo different yet characteristic processing events during their synthesis and export. gp5O (HSV gD homolog) is unusual in that it has 0-linked modifications but no N-linked glycosylation (22). gII (HSV gB homolog) forms oligomers during export, is modified primarily by Nlinked glycosylation, and is also processed by a late Golgi protease (39). gIIl (HSV gC homolog) is highly modified, with both N-linked and probably 0-linked sugars (30). BFA disrupted the kinetics and extent of processing of these three glycoproteins in infected cells but had little effect on their primary synthesis. After BFA treatment, a novel species of gp5O accumulated that was not observed in infected cells. We speculate that since this protein carries only 0-linked modifications, the novel species represents the addition of the 0-linked core sugars in the transitional elements of the ER (36). Since sialyltransferases most likely are located in the trans-Golgi network (36) and since enzymes of this compartment do not gain access to the ER
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FIG. 5. Fine structural morphology typical of PR V-infected cells. The micrograph was made at 8 h postinfection. Infected cells at this time characteristically exhibit pronounced invaginations of the cell nucleus and numerous free capsids in the nucleus (arrows) and cytoplasm. Note the paracrystalline array of capsids in one of the nuclear lobules. N, Nucleus. Bar 500 nm.
during
BFA treatment
0-linked sugars
gpSO
(14), the final modifications
to
the
completed. The block of gll export and processing by BFA treatment is completely consistent with previous models for gll export and processing (39). BFA treatment results in the accumulation of the 100-kDa primary translation product carrying high-mannose modifications. The 110-kDa species, proposed to arise by modification in the Golgi apparatus, is undetected on
are
never
in the presence of BFA. Moreover, the protease-processed
forms proposed to arise in
late
Golgi compartment are not observed. In addition, the oligomerization of the 100-kDaLgII precursor suggested to occur in the ER is still observed after BFA treatment (Fig. 3). a
completely blocked the normal processing of gilll high-mannose glycosylation in the ER. The appearance of partially endoglycosidase H-resistant species 90 min after the pulse in the presence of BFA suggests that the gill precursor is subjected to modifications after prolonged treatment. This probably reflects recycling of specific Golgi enzymes to the ER and the accumulation of unsialylated 0-linked sugars on the BFA
after its cotranslational synthesis and
precursor,
as
observed for
gp5O,
which is consistent with
published data for uninfected cells (13, 15). Enveloped virions in BFA-treated cells are not infectious. Not only does BFA dramatically affect PRV glycoprotein processing and export, but the inhibitor also abruptly stops
VOL. 65, 1991
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the accumulation of infectious intracellular and released particles (Fig. 4). Nevertheless, examination of electron micrographs revealed significant accumulation of enveloped particles in the nuclear envelope and the ER (Fig. 9 and 10). Our conclusion from these results is that virus egress is blocked and that the accumulated intracellular, enveloped particles are noninfectious. We have not yet studied the composition of these particles, so we can only speculate as to why they are not infectious. We expect that they have precursor viral glycoproteins in their envelopes, but it is unclear whether any abnormal modifications due to Golgi enzymes recycling back to the ER as a result of BFA
treatment are present. We know that in BFA-treated cells the essential PRV glycoprotein gll does not form mature oligomers, nor is it proteolytically processed (Fig. 3). It is possible that the particles from BFA-treated cells are noninfectious because gll is nonfunctional. It is well established that the absence of N-linked glycans in HSV glycoproteins resulting from tunicamycin treatment leads to a dramatic reduction in the production of infectious virus (23). In contrast, high yields of infectious HSV are produced in cells which synthesize only immature forms of HSV glycoproteins carrying high-mannose glycans, as in mutant ricinresistant cells (Ric4) (3) or cells treated with monensin (9) or
1074
J. VIROL.
WHEALY ET AL.
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JOURNAL OF VIROLOGY, Mar. 1991, p. 1066-1081 0022-538X/91/031066-16$02.00/0 Copyright C) 1991, American Society for Microbiology
Effect of Brefeldin A on Alphaherpesvirus Membrane Protein Glycosylation and Virus Egress M. E. WHEALY, J. P. CARD, R. P. MEADE, A. K. ROBBINS, AND L. W. ENQUIST* E. I. duPont de Nemours, Central Research and Development, Viral Diseases Group, Experimental Station, Wilmington, Delaware 19880-0328 Received 23 August 1990/Accepted 12 November 1990
In this work we used brefeldin A (BFA), a specific inhibitor of export to the Golgi apparatus, to study pseudorabies virus viral glycoprotein processing and virus egress. BFA had little effect on initial synthesis and cotranslational modification of viral glycoproteins in the endoplasmic reticulum (ER), but it disrupted subsequent glycoprotein maturation and export. Additionally, single-step growth experiments demonstrated that after the addition of BFA, accumulation of infectious virus stopped abruptly. BFA interruption of virus egress was reversible. Electron microscopic analysis of infected cells demonstrated BFA-induced disappearance of the Golgi apparatus accompanied by a dramatic accumulation of enveloped virions between the inner and outer nuclear membranes and also in the ER. Large numbers of envelope-free capsids were also present in the cytoplasm of all samples. In control samples, these capsids were preferentially associated with the forming face of Golgi bodies and acquired a membrane envelope derived from the trans-cisternae. Our results are consistent with a multistep pathway for envelopment of pseudorabies virus that involves initial acquisition of a membrane by budding of capsids through the inner leaf of the nuclear envelope followed by deenvelopment and release of these capsids from the ER into the cytoplasm in proximity to the trans-Golgi. The released capsids then acquire a bilaminar double envelope containing mature viral glycoproteins at the trans-Golgi. The resulting doublemembraned virus is transported to the plasma membrane, where membrane fusion releases a mature, enveloped virus particle from the cell.
Pseudorabies virus (PRV;
a
It is not clear whether these two models are mutually exclusive or are indicative of multiple pathways for envelopment among alphaherpesviruses. To gain insight into this problem, we have examined the pathway for assembly and egress of PRV during the normal course of infection and following treatment with brefeldin A (BFA), an inhibitor of transport between the ER and the Golgi apparatus which ultimately leads to dissolution of the Golgi cisternae. BFA is a potent antiviral agent with marked effects on herpesvirus replication (35). It is now established that BFA disrupts the movement of newly synthesized membrane proteins into the Golgi apparatus (14) and that this effect is reversible (15). After treatment of the cell with BFA, Golgi enzymes are redistributed to the ER, where they remain active (15). Moreover, the Golgi apparatus of BFA-treated cells rapidly disappears as a morphologically distinct entity (14). We reasoned that if the Johnson and Spear model was correct for PRV, then a specific block in ER-to-Golgi apparatus transport should result in the accumulation of enveloped virions in the ER. Furthermore, if the Jones-Grose model was correct for PRV, then an inhibitor that destroys the Golgi apparatus should completely block the formation of intracellular enveloped particles. In this report, we show that BFA has dramatic effects on PRV membrane protein glycosylation, processing, and virus egress consistent with a pathway involving elements of both the HSV and VZV models. Our results are consistent with a multistep pathway for envelopment of PRV that initially involves acquisition of a membrane by budding of capsids into the perinuclear space followed by transport through the ER and deenvelopment, with release of the capsids into the cytoplasm in proximity to the trans-Golgi. The next step involves a second envelopment of these capsids by membrane derived from the transGolgi containing mature, fully processed viral glycoproteins. The resulting double-membraned virus is transported to the
swine pathogen), varicella-
zoster virus (VZV; a human pathogen), and herpes simplex virus type 1 (HSV-1; a human pathogen) are typical mem-
bers of the Alphaherpesvirinae subfamily of herpesviruses (28, 29). The envelope of VZV contains at least five glycoproteins (7), and the envelopes of PRV and HSV-1 contain at least seven virus-encoded glycoproteins (29, 33, 40). The function of the multiple viral glycoproteins, their targeting and proper assembly into the virus envelope, the site of initial virus envelopment, and the pathway of virus egress from infected cells present interesting and as yet only partially understood problems in herpesvirus biology. In general, herpesviruses are unique among enveloped viruses in that the primary site of envelopment of herpes virions is thought to be the inner nuclear membrane (1, 5, 6, 9, 16, 18, 19-21, 24, 33, 34). Johnson and Spear (9) suggested a model for HSV-1 envelopment and viral egress by which HSV-1 virions first were enveloped at the inner nuclear membrane, where they acquired viral glycoproteins lacking Golgi modifications. Subsequently, these "immature" virions were transported to the Golgi apparatus, where precursor glycoproteins were modified as the enveloped particle moved through the Golgi stacks. A contrasting model has been proposed for VZV by Jones and Grose (10), who suggested that VZV capsids were released from the nucleus into the cytoplasm by an unspecified mechanism and subsequently budded into post-Golgi transport vesicles, acquiring a "mature" envelope with fully modified glycoproteins. In this model, the mechanism by which VZV capsids leave the nucleus is unspecified, but virions do not enter the endoplasmic reticulum (ER) nor do they enter the Golgi stacks.
*
Corresponding author. 1066
ENVELOPMENT OF ALPHAHERPESVIRUSES
VOL. 65, 1991
plasma membrane, where fusion releases a mature, enveloped virus particle from the cell.
MATERIALS AND METHODS Materials, cells and viruses. BFA was obtained from Epicentre Technologies, Madison, Wis. It was made up as a 1-mg/ml solution in ethanol and stored at -20°C. The Becker strain of PRV was prepared and used as previously described (26). The cell line used for PRV virus infections was PK15 cells (swine kidney fibroblasts). Growth curves. Single-step growth curves of PRV were done as described by Whealy et al. (38), with modifications as noted in the legend to Fig. 4. Antibody reagents. The antisera used in these studies included a mouse monoclonal antibody, 6D8MB4, reactive with gpSO (a kind gift from C. Whetstone, Ames, Iowa), a goat polyvalent antiserum (no. 282) raised against a denatured Escherichia coli-produced Cro-gIll fusion protein which is reactive with native and denatured glll protein (27), and a goat polyvalent antiserum (no. 284) which recognizes the native and denatured forms of the gII glycoprotein (25). Pulse-chase analysis. The pulse-chase procedure used has been described previously (30). Briefly, PK15 cells were infected at a multiplicity of infection of 10 with PRV Becker. Where indicated, 2.5 ,ug of BFA per ml was added at S h postinfection and was maintained throughout the experiment. The cells were deprived of cysteine at 5.5 h postinfection, and a 2-min radioactive pulse of 100 ,uCi of [35S]cysteine per ml was administered at 6 h postinfection. Radiolabel was then removed, and the cells were incubated in medium containing excess nonradioactive cysteine for various times as indicated in the figure legends. At the desired chase times, monolayers were harvested and samples were immunoprecipitated with specific antiserum. After immunoprecipitation, some samples were digested with the enzyme endoglycosidase H according to the specifications provided by New England Nuclear Corp. All immunoprecipitates were loaded onto sodium dodecyl sulfate (SDS)10% polyacrylamide slab gels. Fluorography was conducted with sodium salicylate (4) and was followed by autoradiography. Measurement of gIl oligomer formation by sedimentation through sucrose gradients. gIl oligomer formation was measured as described by Whealy et al. (39) with modifications as noted in the legend to Fig. 3. Electron microscopy. (i) Sample preparation. Paired samples of BFA-treated and untreated control cells were collected 5.5, 6, 7, and 8 h following infection of cultures with PRV. The protocol for infection of PK15 fibroblast cultures and treatment of cells with BFA reproduced that described for the pulse-chase analysis. At each time point, samples were immersed in a buffered aldehyde solution (5% transmission electron microscopy-grade glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium phosphate buffer [pH 7.4]) and fixed at room temperature for 1 h. Thereafter, samples were washed extensively with buffer prior to and following a 1-h postfixation in buffered 2% osmium tetroxide solution containing 1.5% potassium ferricyanide (13), dehydrated in a graded series of ethanol, and embedded in Epon plastic resin using standard methods. Thin (800-nm) plastic sections were cut perpendicular to the plane of the cell monolayer with a Reichert Ultracut E ultramicrotome. A series of sections was collected on Formvar-coated slot grids, stained with heavy-metal salts (uranyl acetate and lead citrate), and
1067
analyzed with either Hitachi 600 or JEOL 1200ex transmission electron microscopes. (ii) Analysis. Cells from each sample were systematically photographed and examined for the effects of BFA treatment at each time point. As noted above, paired samples of treated and untreated cells were included for each time point and processed in parallel to reduce variations in cellular preservation that might occur in the course of tissue preparation. Analysis of the tissue was conducted in three phases. First, 10 complete cells from each sample were photographed at an intermediate magnification of x 10,000, and photomontages of the cells were assembled at a final magnification of 25,000 diameters. Cells chosen for this analysis exhibited large cross-sectional areas of both the cell nucleus and the cytoplasm. Second, 430 higher-magnification (x 20 to x40,000) micrographs (approximately 50 per sample) were taken of representative areas of cells in each sample. We selected these areas to include long stretches of nuclear envelope and/or organelle-rich regions of cytoplasm. These negatives were enlarged 2.5 times for a final print magnification ranging from xS0 to x100,000. Third, having completed the first two phases of the analysis, we conducted a directed examination in which we photographed those features which were most characteristic of each time point and treatment group. Two aspects of the material were subjected to quantitative analysis, and comparisons were made between experimental and control groups. These included (i) the number of virions per unit membrane of nuclear envelope or ER (rough and smooth) and (ii) the number of viral particles (capsids and enveloped virions) in either the nucleus or the cytoplasm. The first analysis was restricted to linear (longitudinal) stretches of membrane measured with a cartographer's tool. RESULTS BFA does not affect PRV glycoprotein synthesis but blocks processing and export. Previous studies documenting the effects of BFA on cell metabolism and morphology were conducted in the absence of a viral infection. Consequently, we felt it was important to establish basic parameters of the effect of BFA on PRV membrane protein synthesis. This is necessary because PRV, like all alphaherpesviruses, significantly alters host cell metabolism after infection (2). We analyzed the synthesis and export of three well-characterized PRV membrane proteins by pulse-chase analysis. These three glycoproteins, gIl, gIll, and gp5O, were chosen because of specific posttranslational modifications that enabled us to judge the effects of BFA. Briefly, the gIl glycoprotein of PRV contains six N-linked glycosylation sites, forms oligomers in the ER, and, following transport to the Golgi, is processed by a protease (39). The gIll glycoprotein of PRV contains 8 N-linked glycosylation sites that are converted to complex forms in the Golgi, and gIll is likely to be modified by 0-linked glycosylation (30). Finally, the gpSO glycoprotein of PRV has been shown to contain only 0-linked and not N-linked oligosaccharide modifications (22). Infected cells were labeled for 2 min with [35S]cysteine (pulse), and then the isotope was removed and replaced with medium containing an excess of nonradioactive cysteine (chase). Samples were taken at various times and immunoprecipitated with PRV glycoprotein-specific antisera. Identical experiments were done in the presence of BFA. Infected cells were treated with 2.5 ,ug of BFA per ml for 1 h prior to the 2-min pulse. BFA was maintained at 2.5 ,ug/ml during the chase. The results of these experiments are shown
WHEALY ET AL.
1068
J. VIROL.
-BFA 0
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number of gll-related polypeptides. At the 15-min chase a 100-kDa species, representing the completed gIl molecule with high-mannose glycosylation, was apparent. This species was subsequently converted to a 110-kDa species after 45 min of chase. The 110-kDa species began to be processed to the 68- and 55-kDa species by about 45 min after the pulse. In the presence of BFA, the 2-min pulse profile remained essentially identical to that seen in the absence of inhibitor. Therefore, BFA did not affect initial synthesis and glycosylation of gll. However, conversion of the 100-kDa species to the 110-kDa species as well as subsequent protease processing of the 110-kDa species was completely blocked by BFA. The labeling kinetics of gpSO are shown in Fig. 1C. In the absence of BFA, the profile is essentially that reported by Petrovskis et al. (22). We noted that the gp5O monoclonal antibody did not recognize anything synthesized in the pulse, a result obtained with all the gpSO monoclonal antibodies we tested. A similar observation was reported by Petrovskis et al. (22). At 15 min after the pulse, a 46-kDa species was immunoprecipitated, and this form was rapidly converted to a 55-kDa species by 30 min after the pulse. A different profile was observed in the presence of BFA. At 15 min of chase, the 46-kDa protein was replaced by a novel 49-kDa species that was never converted to the 55-kDa form. Novel endoglycosidase H-resistant glll species arise after prolonged treatment with BFA. While BFA completely blocked the formation of predicted Golgi-modified forms of all three PRV glycoproteins, it is obvious that in the presence of BFA, the primary glycosylated species of glIl and gp5O begin to acquire new modifications that retard electrophoretic mobility. These species are never seen in untreated infected cells. This phenomenon is not unexpected. It has been established that BFA treatment of uninfected cells results in rapid redistribution of some Golgi proteins into the ER and that some of the Golgi enzymes retain activity in their new environment (13, 15). We considered the possibility that these forms result from the action of Golgi enzymes redistributed to the ER after BFA-induced dissolution of the Golgi apparatus. One of the Golgi modifications that may be occurring is the conversion of the high-mannose N-linked oligosaccharides to complex forms. This can be tested by measuring the endoglycosidase H sensitivity of the glycoproteins. We concentrated on glll for this analysis. The results are shown in Fig. 2. An aliquot from each of the samples used for Fig. 1A (glll) was digested with endoglycosidase H as described in Materials and Methods. Some aliquots were untreated and some were treated with BFA as described in the legend to Fig. 1A. Ryan et al. (30) demonstrated that the 74-kDa form of gIll was sensitive to endoglycosidase H, resulting in formation of a 58-kDa form, but the 92-kDa species was resistant to digestion. The results in Fig. 2 are essentially identical to these findings. In the presence of BFA (Fig. 2), the 74-kDa precursor was initially sensitive to endoglycosidase H digestion, giving rise to the expected 58-kDa deglycosylated form of gIII. However, as the chase continued in the presence of BFA, modifications appeared that could not be removed by endoglycosidase H. Enzyme digestion reduced the apparent molecular mass, but the final digestion product was larger than the expected 58-kDa deglycosylated form. In other experiments (data not shown), similar samples were also treated with endoglycosidase F, which should cleave both the high-mannose and the complex carbohydrates added to the N-linked sites. This enzyme reduced the apparent molecular mass more than endoglycosidase H treatment but did
point,
-U
.,
_
FIG. 1. Pulse-chase analysis of PRV glycoproteins in the absence and presence of BFA. PK15 cells were infected with PRV at a multiplicity of infection of 10. At 5 h postinfection, BFA was added at a concentration of 2.5 ,ug/ml. The cells were pulse-labeled at 6 h postinfection and chased for the times indicated across the top in minutes. The cells were lysed and immunoprecipitated with gIllspecific serum (A), gll-specific serum (B), or gpSO-specific serum (C). Immunoprecipitates were resolved on a 10% SDS-polyacrylamide gel and visualized by fluorography. The molecular mass standards (in kilodaltons) are indicated to the left of each panel.
in Fig. 1. The left side of each panel contains the untreated controls, and the right side contains BFA-treated cells. Immunoprecipitations of glll, gll, and gpSO are shown. The primary product of glll synthesis was a 74-kDa protein fully glycosylated with high-mannose oligosaccharides (30). Figure 1A demonstrates that by 30 min of chase, the 74-kDa form was converted to a diffusely migrating species with an average apparent molecular mass of 92 kDa known to be resistant to endoglycosidase H and sensitive to endoglycosidase F (30). In the presence of BFA, the 74-kDa form was synthesized, indicating that initial high-mannose glycosylation was unaffected. However, the conversion of the 74-kDa form to the 92-kDa species was completely blocked. At late chase times in the presence of BFA, the 74-kDa species exhibited a slight but reproducible retardation in electrophoretic mobility. The labeling kinetics of glycoprotein gll are shown in Fig. 1B. In the absence of BFA, the profile is that described by Whealy et al. (39). The primary products of synthesis were a
VOL. 65, 1991
ENVELOPMENT OF ALPHAHERPESVIRUSES -BFA
0
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i20 0
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045
on
6
FIG. 2. Endoglycosidase H treatment of PRV glll. PK15 cells infected with PRV at a multiplicity of infection of 10. BFA (2.5 ,ug/ml) was added at 5 h postinfection where indicated. At 6 h postinfection, the cells were pulse-labeled and chased for the times indicated (in minutes). The cells were lysed and immunoprecipitated with gIll-specific serum. The immunoprecipitates were treated overnight with endoglycosidase H as described by the manufacturer prior to fractionation on a 10% SDS-polyacrylamide gel and visualization by fluorography. The molecular mass standards (in kilodaltons) are indicated to the left. were
not reduce it to that of the expected 58-kDa deglycosylated form. This indicates that there is some conversion of the high-mannose N-linked oligosaccharides to complex forms, presumably by Golgi enzymes that have recycled back to the ER after BFA treatment. In addition, other modifications, such as 0-linked sugars, may be added in the presence of BFA. Formation of primary gIl oligomers is not blocked by BFA treatment. Whealy et al. (39) demonstrated that gIl is initially synthesized in the ER as a monomer with high-mannose modifications. This 100-kDa precursor is converted to an oligomer (most likely a homodimer) by about 30 min. Subsequently, the precursor oligomer is converted to an oligomer of identical sedimentation value but composed of 110-kDa monomers by the partial conversion of the highmannose sugars to complex forms in the Golgi apparatus. Next, the mature oligomer is cleaved by a cellular endoprotease, resulting in a complex with the subunits held together both by disulfide and SDS-resistant interactions (39). On the basis of kinetics of export and processing (39), the cellular protease was deduced to be a late Golgi enzyme. In the first section, we determined that BFA blocked the conversion of the 100-kDa precursor to the 110-kDa species and that no protease processing of the precursor occurred. It was of interest to determine whether the 100-kDa monomers had been converted to oligomers. To determine this, infected cells were treated with 2.5 ,ug of BFA per ml at 5 h postinfection. At 6 h postinfection, cells were labeled for 2 min with [35S]cysteine and then chased for 10 and 60 min with excess cold cysteine as described by Whealy et al. (39). The 10- and 60-min chase samples were solubilized in Triton X-100 and fractionated on sucrose gradients as described by Whealy et al. (39). Gradient fractions were immunoprecipitated with anti-gIl serum, and the precipitates were analyzed on an SDS-polyacrylamide gel (Fig. 3). It is clear that in the presence of BFA, the gII highmannose-modified precursor of 100 kDa sedimented as a monomer at the 10-min chase point and by 60 min after synthesis was converted into oligomers cosedimenting with normal gII oligomers. These data support the suggestion of Whealy et al. (39) that primary oligomerization of gII occurs in the ER prior to export to the Golgi apparatus.
1069
BFA blocks formation of infectious particles as determined by single-step growth experiments. It was clear from the experiments described above that BFA had a dramatic effect on PRV glycoprotein maturation and export. As reported by others for uninfected cells, BFA caused a specific and essentially complete block of ER-to-Golgi apparatus transport as measured by lack of complex N-linked-sugar formation, completion of 0-linked-sugar modifications, and proteolytic processing of viral glycoprotein oligomers. Therefore it was of interest to determine the effects of BFA on virus particle formation. We first examined the effects of BFA on formation of infectious particles. For this experiment, a series of plates was infected at a multiplicity of infection of 5 and incubated at 37°C. At 1, 3, 5, 7, 9, 11, 13, and 24 h postinfection, infected cells and media were harvested and virus titers of the cell-associated and medium fractions were determined. At 5 h postinfection, 2.5 ,ug of BFA per ml was added to one set of plates. The results of this experiment are shown in Fig. 4A. In the absence of BFA, results were essentially identical to those described previously for this strain of PRV (38). However, upon the addition of BFA, production of infectious virus stopped abruptly, with the number of infectious particles remaining relatively constant for the duration of the experiment. The infectious virus remaining after BFA treatment must represent extracellular particles formed prior to the addition of BFA which adhere to the extracellular matrix since they are sensitive to low-pH citrate treatment (data not shown). This treatment is known to kill PRV particles outside cells but has no effect on virions inside the cell (17). A second experiment was done to determine whether the effects of BFA on infectious-particle formation were reversible. A single-step growth experiment was done as described above except that after 30 min of BFA treatment at 5 h postinfection, the drug was removed and replaced with drug-free medium for the duration of the experiment. Infected cells were scraped into the medium at 30-min intervals after the addition of BFA, and titers were determined for plaque-forming particles (Fig. 4B). As shown in Fig. 4A, the addition of BFA blocked the accumulation of infectious particles. After the removal of BFA, a lag of about 1 h ensued and then infectious particles began to accumulate at a rate similar to that of the untreated control. By 9 h postinfection, both treated and untreated cultures accumulated similar titers of infectious virus. We conclude that the effects of BFA on infectious-particle formation are reversible. Morphological consequences of PRV infection and effects of BFA treatment. BFA clearly stopped the formation of infectious virus, but at what stage were virus assembly and egress blocked? We used transmission electron microscopy to give us insight into this problem. To appreciate more fully the effects of BFA on PRV-infected cells, it was important to establish basic morphological parameters in infected but untreated cells. In what follows, representative electron micrographs will be referred to, but the conclusions are never drawn from a single micrograph. Specifically, at least 10 complete cells from each time point (5.5, 6, 7, and 8 h after infection) were examined, and each examination involved more than 50 higher-magnification micrographs of representative areas of the cell. The details of our analysis are described in Materials and Methods. Figure 5 illustrates the major morphological alterations that occurred in PK15 cells in response to PRV infection. Shown is a typical infected cell at 8 h after infection. Pathological invaginations of the nuclear envelope, disas-
1070
J. VIROL.
WHEALY ET AL. Fractions Dimer 2
3
4
5
6
7
8
9
10
11
Monomer 12
13
14
15
16
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18
19
10' Chase
_
___
_
92 -
68
46 -
60' Chase
92 -
68 -
46 -
FIG. 3. Gradient fractionation of PRV gll. PK15 cells were infected at a multiplicity of infection of 10. BFA was added at a concentration of 2.5 ,ug/ml at 5 h postinfection. At 6 h postinfection, the cells were pulse-labeled and chased for 10 or 60 min as indicated. The cells were solubilized in 1% Triton X-100 and fractionated by sedimentation through a 5 to 15% sucrose gradient containing 0.1% Triton X-100. The fractions were immunoprecipitated with a gll-specific antiserum. The immunoprecipitates were resolved on a 10% SDS-polyacrylamide gel and visualized by fluorography. The positions of the molecular mass standards (in kilodaltons) resolved on the polyacrylamide gel are indicated to the left of each panel. The relative positions of the expected monomers and dimers of gll are indicated at the top.
sembly of the nucleolus, and clumping of chromatin were the hallmarks of infection. In addition, infected cells characteristically exhibited pronounced accumulations of capsids both in the nucleus and the cytoplasm (Fig. 5). Moreover, capsids in various stages of envelopment were commonly associated with the forming face of the Golgi apparatus. These morphological indicators increased dramatically as infection proceeded. For example, most cells at 5.5 h postinfection exhibited only a few fingerlike invaginations in the nuclear envelope, minimal clumping of the nuclear chromatin, and a moderate number of nucleocapsids dispersed throughout the nucleoplasm. However, by 8 h postinfection, multiple irregular nuclear invaginations analogous to those illustrated in Fig. 5 characterized the majority of infected cells, and large crystalline arrays of nucleocapsids typically were present adjacent to the nuclear envelope. It is important to note that only in rare instances did we observe virions between the inner and outer leaflets of the nuclear envelope. The cytoplasm contained a variety of morphologically distinct virus particles that accumulated as the infection proceeded. We noted that these particles exhibited a preferential association with the Golgi cisternae. Although individual virions occurred throughout the cytoplasm, the majority of particles were concentrated in focal aggregations immediately adjacent to the trans cisternae of the Golgi apparatus (Fig. 6). These areas characteristically consisted of a com-
plex array of capsids, immature virions, and bilaminar membrane profiles. Rough ER membranes were prevalent in the vicinity of these areas and in some instances were found in the midst of the virion aggregations (Fig. 6, 7a, and 8). However, most membranous profiles had no associated ribosomes and in many instances were continuous with the trans-cisternae of the Golgi apparatus (Fig. 6 and 7a). These membranous arrays surrounded individual capsids and ultimately fused to form a bilaminar envelope (Fig. 6 and 7). The resulting "virions" were composed of central capsids surrounded by two distinct membranes, with an average crosssectional diameter of approximately 200 nm. To confirm that capsids were principally associated with the trans-cisternae of the Golgi apparatus, we examined serial thin sections through the regions of virion aggregation (Fig. 8). This analysis demonstrated that several Golgi complexes were associated with an individual aggregation. This is clearly evident in the consecutive sections illustrated in Fig. 8: seven different Golgi complexes are intimately associated with a single prominent region of capsid aggregation immediately adjacent to the cell nucleus. With the exception of one complex (no. 2), all of the Golgi bodies were found at the periphery of the capsid aggregation. In addition, the trans-cisternae of all of the profiles are oriented toward the virion aggregation, and there are a number of clear examples of extensions of the trans-cisternae sur-
1991
VOL. 65,
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ENVELOPMENT OF ALPHAHERPESVIRUSES
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FIG. 4. Single-step growth curves of PRV. PK] infected with PRV at a multiplicity of infection of 5 an d incubated at 370C. (A) At 1, 3, 5, 7, 9, 11, 13, and 24 h postinfectio n, plates were harvested and the virus titers of the cell (0) and me dium fractions (Q) were determined separately. At S h postinfecti ion, BFA (2.5 mg/ml) was added to a duplicate set of plates (-) ancJ harvested as described in the text for untreated cells. Data for BFA-treated cell-associated samples (-) and medium samples (LI) a3re shown. (B) At 1 and 3 h postinfection, the infected cells were sciraped into the medium and titers were determined. At 5 h postinfecti ion, half of the plates were treated with BFA at a concentration of 2.5 j±gIml (T).At 5.5 h postinfection, the medium was removed and disc arded from all plates, the monolayers were washed with phosphate-b uffered saline, and fresh Dulbecco modified Eagle medium was ad led (O). Cells were harvested at 5, 5.5, 6, 6.5, 7, 7.5, and 9 h po stinfection as described in the text for 1 and 3 h (&, no BFA; A 30-min BFA treatment). The scale on the left is the log of the p laque-forming units per milliliter.
rounding individual capsids (see Golgi complex no. 3 in Fig. 8a and b). BFA treatment resulted in rapid dissolution of the Golgi cisternae and a dramatic accumulation of viri(cns between the inner and outer nuclear membranes as well aas in the ER. Other than loss of the Golgi structures and acciumulation of enveloped virions, no alterations in cellular morphology were visible. These effects were evident withiin 30 min of application of the drug and became more dramattic with time. In all instances, prominent accumulations of virions were apparent between the internal and external le aflets of the nuclear envelope (Fig. 9a and b and 10a thr ough c) and within the rough ER of the cytoplasm (Fig. 9c). Quantitation of the number of virions in the nuclear envelc pes and the
1071
rough ERs of control and experimental samples at each time point provided a striking illustration of this effect (Fig. 11). At 30 and 60 min following BFA treatment, we saw no difference in the number of virions per 10 ,um of membrane in either the nuclear envelope (Fig. 11A) or the ER (Fig. 11B). However, at 2 and 3 h after BFA treatment, we observed a marked increase in the number of virions in each of these compartments that was not apparent in paired control samples. The number of virions in the nuclear envelope increased from approximately 0.5 virions per 10 ,um of membrane at 30 min post-BFA treatment to more than 7 virions per 10 ,um at 3 h posttreatment (Fig. 11A). A similar but less dramatic increase occurred in the ER (Fig. 11B). Since the linear extent of ER far exceeds that of the nuclear membrane, this is a predicted result. These differences occurred even though the relative number of capsids and enveloped virions per cell remained relatively constant in both untreated and BFA-treated infected cells (data not shown). The morphology of virions apparent between the inner and outer nuclear membranes of BFA-treated cells varied considerably. We observed structures containing profiles of relatively immature capsids surrounded by a single membrane to capsids surrounded by a prominent tegument and a dense envelope reminiscent of that observed for mature virus in untreated cells. Numerous micrographs showed that the less-mature form of virion acquired its membrane by budding through the inner nuclear membrane (Fig. 10a and b). Similarly, we observed several examples of capsids gaining access to the cytoplasm by fusion of this membrane with the outer leaf of the nuclear envelope (Fig. 10c). This observation was consistent with the presence of numerous free capsids within the cytoplasm of BFA-treated samples. However, the location and organization of these free cytoplasmic capsids differed from those observed in paired controls. In essentially every case, most capsids observed in the cytoplasm of BFA-treated cells were concentrated around spherical accumulations of dense granular material (Fig. 10d). These granular matrices were more commonly observed in BFA-treated cells and appeared to arise coincident with the dissolution of the Golgi cisternae. DSUSO DISCUSSION
BFA blocks PRV glycoprotein processing and export. BFA dramatically effected PRV membrane glycoprotein processing and export. We examined three unique viral membrane glycoproteins that undergo different yet characteristic processing events during their synthesis and export. gp5O (HSV gD homolog) is unusual in that it has 0-linked modifications but no N-linked glycosylation (22). gII (HSV gB homolog) forms oligomers during export, is modified primarily by Nlinked glycosylation, and is also processed by a late Golgi protease (39). gIIl (HSV gC homolog) is highly modified, with both N-linked and probably 0-linked sugars (30). BFA disrupted the kinetics and extent of processing of these three glycoproteins in infected cells but had little effect on their primary synthesis. After BFA treatment, a novel species of gp5O accumulated that was not observed in infected cells. We speculate that since this protein carries only 0-linked modifications, the novel species represents the addition of the 0-linked core sugars in the transitional elements of the ER (36). Since sialyltransferases most likely are located in the trans-Golgi network (36) and since enzymes of this compartment do not gain access to the ER
1072
WHEALY ET
J. VIROL. AL.J.VRL
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FIG. 5. Fine structural morphology typical of PR V-infected cells. The micrograph was made at 8 h postinfection. Infected cells at this time characteristically exhibit pronounced invaginations of the cell nucleus and numerous free capsids in the nucleus (arrows) and cytoplasm. Note the paracrystalline array of capsids in one of the nuclear lobules. N, Nucleus. Bar 500 nm.
during
BFA treatment
0-linked sugars
gpSO
(14), the final modifications
to
the
completed. The block of gll export and processing by BFA treatment is completely consistent with previous models for gll export and processing (39). BFA treatment results in the accumulation of the 100-kDa primary translation product carrying high-mannose modifications. The 110-kDa species, proposed to arise by modification in the Golgi apparatus, is undetected on
are
never
in the presence of BFA. Moreover, the protease-processed
forms proposed to arise in
late
Golgi compartment are not observed. In addition, the oligomerization of the 100-kDaLgII precursor suggested to occur in the ER is still observed after BFA treatment (Fig. 3). a
completely blocked the normal processing of gilll high-mannose glycosylation in the ER. The appearance of partially endoglycosidase H-resistant species 90 min after the pulse in the presence of BFA suggests that the gill precursor is subjected to modifications after prolonged treatment. This probably reflects recycling of specific Golgi enzymes to the ER and the accumulation of unsialylated 0-linked sugars on the BFA
after its cotranslational synthesis and
precursor,
as
observed for
gp5O,
which is consistent with
published data for uninfected cells (13, 15). Enveloped virions in BFA-treated cells are not infectious. Not only does BFA dramatically affect PRV glycoprotein processing and export, but the inhibitor also abruptly stops
VOL. 65, 1991
ENVELOPMENT OF ALPHAHERPESVIRUSES A..
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the accumulation of infectious intracellular and released particles (Fig. 4). Nevertheless, examination of electron micrographs revealed significant accumulation of enveloped particles in the nuclear envelope and the ER (Fig. 9 and 10). Our conclusion from these results is that virus egress is blocked and that the accumulated intracellular, enveloped particles are noninfectious. We have not yet studied the composition of these particles, so we can only speculate as to why they are not infectious. We expect that they have precursor viral glycoproteins in their envelopes, but it is unclear whether any abnormal modifications due to Golgi enzymes recycling back to the ER as a result of BFA
treatment are present. We know that in BFA-treated cells the essential PRV glycoprotein gll does not form mature oligomers, nor is it proteolytically processed (Fig. 3). It is possible that the particles from BFA-treated cells are noninfectious because gll is nonfunctional. It is well established that the absence of N-linked glycans in HSV glycoproteins resulting from tunicamycin treatment leads to a dramatic reduction in the production of infectious virus (23). In contrast, high yields of infectious HSV are produced in cells which synthesize only immature forms of HSV glycoproteins carrying high-mannose glycans, as in mutant ricinresistant cells (Ric4) (3) or cells treated with monensin (9) or
1074
J. VIROL.
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