VIROLOGY
191,327-337
Studies
(1992)
Complex Cycling Pathway on Endoplasmic Reticulum -Golgi Virus-Infected and Brefeldin A-Treated Human Fibroblast
in Herpes Cells’
Simplex
SUBHENDRA CHAlTERJEE2 AND SAMITA SARKAR Department
of Pediatrics,
BHSB 996, University
of Alabama at Birmingham,
Birmingham,
Alabama 35294
Received May 6, 1992; accepted July 29, 1992 Brefeldin A (BFA), a fungal metabolite, significantly inhibited the release of herpes simplex virus type 1 (HSV-1) from infected human fibroblast cells. Electron micrographs of HSV-l-infected and BFA-treated human cells demonstrated the presence of enveloped particles trapped between outer and inner nuclear membranes. Analyses of viral glycoproteins B, C, and D (gB, gC, and gD) showed faster migrating, immature forms in BFA-treated cells when compared to the mature glycoproteins, as observed in the untreated control cells. The shift in mobilities of the glycoproteins in BFAtreated cells apparently was due to the disassembly of the Golgi complexwhen evaluated by an indirect immunofluorescence assay. The immature forms of gB, gC, and gD could not be detected on the surface of BFA-treated human fibroblast cells. Removal of BFA resulted in a reorganization of the Golgi complex and formation of fully glycosylated gB, gC, and gD. Moreover, the HSV-1 particles released from the treated cells after the removal of BFA completely restored the infectivity of the viral particles. Our results indicate that human fibroblast cells have an endoplasmic o 1992 Academic press, hc reticulum-Golgi cycling pathway.
perinuclear space (Spear, 1985). The virion glycoproteins are processed through the Golgi complex and converted from the “high-mannose type” (i.e., immature form) to the “complex type” (i.e., mature form). Recent studies demonstrated that brefeldin A (BFA), a fungal metabolite, blocked the transport of vesicular stomatitis virus (VSV) G protein from the ER to the Golgi complex (Doms et a/., 1989; Misumi et al., 1986; Takatsuki and Tamura, 1985). In Punta Toro virus, Chen et a/. (1991) reported that both the Gl and G2 proteins were transported out of the ER and retained in the Golgi complex. In pseudorabies virus, Enquist’s group demonstrated that although BFA had little effect on initial synthesis and modification of viral glycoproteins in the ER, it disrupted subsequent glycoprotein maturation and export (Whealy et al., 1991). Although the exact mechanism of action is unknown, it has been reported that BFA causes intracellular redistribution of the Golgi complex, leaving no identifiable structure (Doms et al., 1989; Lippincott-Schwartz et al., 1990). It has also been reported that BFA prevents the transport of HSV-1 particles from the perinuclear space into the extracellular media and the effects of BFAon HSV propagation are not fully reversible (Cheung et al., 1991). In this report, we demonstrate that BFA treatment (as little as 0.05 pg per milliliter) led to the synthesis of partially glycosylated gB, gC, and gD, probably due to the disassembly of Golgi complex. These partially glycosylated, immature forms of gB, gC, and gD were not detectable on the surface of BFA-treated, HSV-1 -infected cells. Electron micrographs of HSV-1 -infected, BFA-
INTRODUCTION Membrane glycoproteins of enveloped animal viruses are, in general, synthesized, processed, and transported inside infected cells in a sequential manner. While the intracellular transport and expression of viral glycoproteins on the cell surface are essential for many biological functions, the details of this transport pathway are poorly understood. In herpes simplex virus type 1 (HSV-1) infected cells, at least nine glycoproteins, gB, gC, gD, gE, gG, gH, gl, gK, and gL (Baucke and Spear, 1979; Eberle and Courtney, 1980; Gompels and Minson, 1986; Hutchinson et a/., 1991; Longnecker et al., 1987; Marsden et a/., 1984; Roizman et a/., 1984) have been identified. The synthesis and transport of HSV-1 glycoproteins have been studied by several investigators (Chatterjee and Burns, 1990; Chatterjee et al., 1990; Glorioso et al., 1983; Johnson and Spear, 1982; Johnson and Spear, 1983; Norrild and Pedersen, 1982) but the exact pathway for processing and transport of these glycoproteins is unclear. Viral glycoproteins, which are synthesized in the endoplasmic reticulum (ER) are transported to the nuclear membrane in an endo Hsensitive form and assembled into the virions (Compton and Courtney, 1984). These viruses bud into the
’ A portion of this investigation was presented at the General Meeting of the American Society for Microbiology, May 5-9, 1991, in Dallas, Texas. ’ To whom reprint requests should be addressed. 327
0042-6822/92
$5.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction I” any form reserved
CHAlTERJEE
328
treated cells demonstrated the presence of enveloped particles between the outer and inner nuclear membranes. The results indicate that BFA blocked intracellular processing of HSV-1 glycoproteins and migration of viral particles from the nuclear membranes to the cell surface. However, removal of BFAfrom the culture medium resulted in a reorganization and redistribution of Golgi complex and fully glycosylated gB, gC, and gD. Furthermore, the HSV-1 particles released from the treated cells following the removal of BFA completely restored the infectivity in tissue culture cells. The data indicate that BFA might be useful for studying the intracellular trafficking of glycoproteins and may provide information regarding the reformation and redistribution of cellular organelles. MATERIALS
AND METHODS
Cells and viruses African green monkey kidney (BS-C-1) cells were obtained from the American Type Culture Collection, Rockville, MD, and grown in medium 199 supplemented with 10% heat-inactivated fetal calf serum and gentamicin (50 pg/mI). Human foreskin (Hfs) fibroblast cells were prepared according to published procedures (Paul, 1970). These cells were grown in Dulbecco’s modified Eagle’s medium also supplemented with 10% heat-inactivated fetal calf serum and gentamicin (50 pglml). The F strain of HSV-1 was provided by B. Roizman, The University of Chicago, Illinois. Reagents
and radioisotopes
Brefeldin A was purchased from Epicentre Technologies (Madison, WI). Reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories (Richmond, CA). Nalz51 (13 mCi/pg of iodine) was purchased from Amersham Corp. (Arlington Heights, IL). Protein A from Staphylococcus aureus was purchased from Pharmacia Laboratories (Uppsala, Sweden). Monoclonal antibodies against gB, gC, and gD were prepared in this laboratory (Koga et al., 1986). Rabbit anti-mouse IgG was obtained from Organon Teknika Corp. (West Chester, PA). Fluorescein-conjugated mouse IgG was purchased from Hyclone Laboratories, Inc. (Logan, UT). Rhodamine-conjugated wheat germ agglutinin was purchased from E-Y Laboratories, Inc. (San Mateo, CA). Electron
microscopy
In brief, HSV-l-infected BS-C-1 cells, grown in 60mm-diameter dishes, were treated with different concentrations of BFA. One set of cells served as untreated control. Samples were then carefully washed
AND SARKAR
with phosphate-buffered saline (PBS) 24 hr postinfection and fixed with 1% glutaraldehyde. Fixed cells were postfixed with 1% osmium tetroxide and processed for electron microscopy, as described previously (Chatterjee et al., 1982). Polyacrylamide immunoblotting
gel electrophoresis
and
Untreated and BFA-treated cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9.5%) after infection with HSV-1 (Chatterjee et a/., 1982, 1985). The fractionated proteins were electrophoretically transferred to nitrocellulose paper and processed for immunoblotting (Chatterjee et a/., 1985; Johnson et al., 1984; Towbin et a/., 1979). Nitrocellulose strips were further incubated at room temperature for 30 min with rabbit anti-mouse IgG. After immune reaction, the nitrocellulose strips were treated with [‘251]protein A for 1 hr at room temperature and then processed by autoradiography. Protein A iodination was performed as described by Greenwood et al. (1963). 1251-labeled protein A binding
immunoassay
The procedure for the protein A binding immunoassay was published previously (Chatterjee et a/., 1981, 1989). In brief, human fibroblast cells, seeded onto a 96-well microtiter plate, were treated with different concentrations of BFA after infection with HSV-1. One set of cells served as a control. The monolayers of treated and untreated cells were washed thoroughly with PBS and treated with PBS containing 0.1% bovine serum albumin (PBS-BSA) for 30 min at room temperature. After incubation with PBS-BSA, antibodies to gB, gC, or gD were added to the cells for 1 hr at room temperature. The cells were washed with PBS-BSA buffer and 5 X 1O4 cpm (approximately) of ‘251-labeled protein A was added to each well for 1 hr at room temperature. Finally, the wells were washed with PBSBSA and air dried. The viral antigen-positive wells were detected by autoradiography, as described before (Chatterjee et a/., 1981, 1989). Indirect
immunofluorescence
assay
An indirect immunofluorescence assay of virus-infected, untreated, and virus-infected, BFA-treated cells was performed in four-chamber tissue culture slides as described (Chatterjee and Burns, 1990; Chatterjee and Hunter, 1979). In brief, cells were washed with PBS and fixed in a mixture of 95% ethyl alcohol and 5% glacial acetic acid at -20” for 1 hr. The samples were treated with PBS-BSA for 30 min and then incubated with antibodies to gB or gC or gD for 1 hr at room
ENDOPLASMIC
RETICULUM-GOLGI
TABLE 1
0 0.01 0.05 0.1 0.5
No. of PFU/ml 1.0 2.0 1.0 2.0 1.0
x x x x x
106 lo4 lo2 10' 10'
CYCLING
PATHWAY
Effect of BFA on the assembly HSV-1 nucleocapsids
EFFECTOF BFA ONTHERELEASEOFINFECTIOUS HSV-1 FROM HUMAN FIBROBLASTCELLS' Concentration (rdml)
COMPLEX
% Inhibition 0 98.0 99.9 99.9 99.9
a Human fibroblast cells were infected with strain F of HSV-1 and then treated with different concentrations of BFA for 18 hr. One set of cells served as untreated control. Supernatants from treated and untreated cells were then tested for their ability to form plaques in BS-C-1 cells.
temperature. Cells were washed three times with PBS-BSA and stained with fluorescein-conjugated mouse IgG. After incubation for an additional 1 hr at room temperature, cells were washed thoroughly with PBS-BSA. Finally, cells were treated with rhodamineconjugated wheat germ agglutinin for 30 min at room temperature in order to stain the Golgi complex. Cells were examined by episcopic fluorescence using a Nikon Fluorophot microscope.
RESULTS
329
and budding
of
The intracellular status of HSV-1 nucleocapsids was determined with regard to assembly and budding in BFA-treated cells. Untreated and BFA-treated human fibroblast cells were processed for electron microscopy after HSV-1 infection. Electron micrographs demonstrated HSV-1 nucleocapsids inside the nuclei of both untreated and BFA-treated cells even at a concentration of 0.1 fig/ml (Fig. 1). In addition to the nucleocapsids, enveloped HSV-1 particles were also seen in BFA-treated cells but were restricted to the space between outer and inner nuclear membrane (Fig. 1B, 1C). It was noticed that the space between the outer and inner nuclear membrane became enlarged in BFA-treated cells. No distinct Golgi structure was observed inside the BFA-treated ceils when compared to that observed with the untreated cells (Fig. 1A). Intracellular viral infectivity of the BFA-treated cells was significantly reduced as determined by a plaque assay. In brief, human fibroblast cells were infected and treated with 0.05 and 0.1 pg/ml of BFA for 24 hr. Treated and untreated cells were washed twice and collected in 1 ml of growth medium. Cells were sonicated for 30 set and tested for their ability to form plaques in BS-C-l cells. The result of this experiment showed a significant reduction in the synthesis of infectious particles inside the BFA-treated (0.05 pg/ml) cells (Table 2, upper panel).
Effect of BFA on the release of HSV-1 from human fibroblast cells To determine the dose-response effects of BFA, human fibroblast cells were infected with F strain of HSV1 (multiplicity of infection = 5) for 1 hr and then treated with 0.01, 0.05, 0.1, and 0.5 pg/ml of BFA. One set of cells served as untreated control. Supernatant fluids from treated and untreated cells were collected 18 hr postinfection and tested for their ability to form plaques in BS-C-1 cells. Even 0.01 pglml of BFA significantly blocked the release of infectious HSV-1 from human fibroblast cells (Table 1). These results were confirmed by i,mmuno dot blot evaluation of extracellular viral particles. In brief, culture supernatants collected from BFA-treated and untreated cells 18 hr postinfection were clarified and the virus was pelleted by centrifugation at 40,000 rpm for 1 hr. The pellets were processed for a dot blot assay using rabbit antiserum to HSV-1 in order to determine the total quantity of extracellular viral particles released from treated and untreated cells. A reduction of 97.25% in the quantity of total extracellular viral particles released from BFA-treated (0.05 pg/ml) cells was noticed.
Expression
of viral glycoproteins
in BFA-treated
cells
Since enveloped viral particles were observed within BFA-treated cells, the status of the selected glycoproteins in treated cells was determined. Control and BFA-treated (0.05 and 0.1 pg/ml) cell lysates collected 18 hr postinfection were analyzed by immunoblot technique to assess the expression of gB, gC, or gD using monoclonal antibodies. As displayed in Fig. 2, gB, gC, and gD from BFA-treated cells migrated faster than that observed in the untreated control cells. Thus, the glycoproteins expressed in the presence of BFA appeared only partially glycosylated. In parallel, we also examined the incorporation of glycoproteins into the extracellular viral particles (approximately 3.4 and 2%) released from BFA-treated (0.05 and 0.1 pg per milliliter, respectively) cells. Figure 3 shows that the expression of HSV-1 glycoproteins on the extracellular virus particles produced from BFA-treated cells was completely inhibited. The quantities of extracellular virus particles released from BFA-treated cells were determined by incubating the same nitrocellulose blot
330
CHAlTERJEE
AND SARKAR
FIG. 1. Electron micrographs of HSV-1 particles in BFA-treated and untreated human fibroblast cells. (A) Untreated cell. Distinct inner and outer nuclear membranes and Golgi complex (G) can be observed. Extracellular and intracellular virus particles(V) can also be seen. Magrt ification, X 53,600. (B) Cells treated with BFA (0.1 pg/ml) showing virus particles trapped between the outer and inner nuclear membrane (ar‘rowheads). No distinct Golgi complex was observed. Magnification, X 19,680. (C) Magnified view of a BFA (0.1 pglml) treated cell. Note the enlar -ged perinuclear area with several enveloped virus particles trapped inside. Magnification, X 88,000.
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COMPLEX
FIG. 1 -Continued
CYCLING
PATHWAY
331
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CHATTERJEE AND SARKAR
A
TABLE 2 INFECTIVITYOF THE INTRACELLULARVIRUS PARTICLESIN THE CONTINUOUS PRESENCEOF BFA AND AFTERTHE BFA IS REMOVED Time of treatment 0-d In presence O-24
of BFA
After removal of BFA o-12
with rabbit antiserum counting.
123
No. of PFU/ml
Concentration 0 0.05 0.1
7.0 x lo8 3.0 x 10’ 2.0 x 10’
0 0.05 0.1
7.0 x lo8 7.1 x 106 6.9 x lo6
to HSV-1 followed
Localization of HSV-1 glycoproteins human fibroblast cells
123
B
by gamma
in BFA-treated
Since gB, gC, and gD were expressed in the BFAtreated cells, albeit improperly, we localized these glycoproteins within the BFA-treated cells in order to define the block in glycosylation. Control and BFA-treated (0.1 pg/ml), HSV-1 -infected cells were examined by indirect immunofluorescence as shown in Fig. 4. The result of this experiment demonstrated that BFA caused disassembly of the Golgi complex, leaving no definable structure as evident from the staining pattern with rhodamine-conjugated wheat germ agglutinin
A
B
C
123
123
123
FIG. 3. Cells were infected with strain F and then treated with different concentrations of BFA for 18 hr. One set of cells served as untreated control. Culture supernatants were collected, clarified, and the virus was pelleted by centrifugation at 40,000 rpm for 1 hr. The pellets werefysed and equal amounts (50 ~1)from untreated and treated samples were subjected to polyacrylamide gel electrophoresis and immunoblotting. The nitrocellulose blot was reacted with monoclonal antibodies to gB, gC. and gD. (A) The strip was incubated with monoclonal antibodies to gB and gD. (B) The strip was incubated with monoclonal antibodies to gC. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter: lane 3. 0.1 pg of BFA per milliliter.
(Fig. 4C). In contrast, a distinct Golgi complex could be observed in untreated control cells (Fig. 4A). It is possible that this defect in Golgi complex was responsible for altered or diffused patterns and reduced expression of gC in BFA-treated human cells as evident from the fluorescein-labeled cells (Fig. 4D). Although some cells apparently displayed normal staining patterns, the glycoproteins in these cells were also partially glycosylated as no mature glycoproteins were observed in the immunoblot profiles. A similar pattern of immunofluorescence was observed when antibodies against gB and gD were used instead of antibodies to gC (data not shown). Expression of HSV-1 glycoproteins on the surface of BFA-treated human fibroblast cells
FIG. 2. Expression of HSV-1 gB, gC, and gD in BFA-treated and untreated human fibroblast cells. (A) The strip was incubated with monoclonal antibody to gB. (B) The strip was incubated with monoclonal antibody to gC. (C) The strip was incubated with monoclonal antibody to gD. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter; lane 3, 0.1 rg of BFA per milliliter.
Although expression of gB, gC, and gD was observed in BFA-treated cells, it was not possible from the above experiments to determine whether these glycoproteins were expressed on the surface of BFAtreated cells. To detect the presence of gB, gC, and gD on the surface of BFA-treated cells, ‘251-labeled protein A binding immunoassay was performed. As shown in Fig. 5, 0.1 pg/ml of BFA significantly inhibited the ex-
ENDOPIASMIC
RETICULUM-GOLGI
COMPLEX
CYCLING
333
PATHWAY
FIG. 4. Indirect immunofluorescence assay of BFA-treated and untreated human cells after HSV-1 infection. All samples were reacted with monoclonal antibody to gC. (A) Untreated fluorescein-labeled cells reacted with rhodamine-conjugated wheat germ agglutinin. (B) Untreated cells, fluorescein labeled. The arrows indicate the positions of Golgi complexes. (C) Cells treated with BFA (0.1 pg/ml), fluorescein labeled, and reacted with rhodamine-conjugated wheat germ agglutinin. (D) Cells treated with BFA (0.1 pg/ml), fluorescein labeled. The arrows indicate the positions of disassembled Golgi complexes.
pression of gB, gC, and gD on the surface of infected cells. Structural reorganization removal of BFA
of treated
cells after
To determine whether these effects of BFA were reversible after removal of the drug from the culture me1 2 3 ABC
DE
F
GH
I
FIG. 5. Autoradiogram of 1251-labeled protein A binding assay to demonstrate the expression of HSV-1 glycoproteins on the surface of BFA-treated and untreated human fibroblast cells. Rows A, B. C, cells incubated with monoclonal antibody to gC. Rows D, E, F. cells incubated with monoclonal antibody to gD. Rows G, H, I, cells incubated with monoclonal antibody to gB. Rows, A, D, G, untreated cells. Rows B, E, H, cells treated with 0.1 rg of BFA per milliliter. Rows C, F, I, cells treated with 0.5 fig/ml of BFA per milliliter. Wells in row 1, uninfected control cells: wells in row 2, cells infected with 2 PFU/cell; wells in row 3, cells infected with 0.5 PFU/cell.
dia, HSV-1 -infected human fibroblast cells were treated with BFA at a concentration of 0.05 or 0.1 pg per milliliter. Twelve hour postinfection BFA containing medium was replaced by growth medium without BFA. Ceils were then processed for electron microscopy 24 hr postinfection as described under Materials and Methods. The resulting electron micrograph (Fig. 6) demonstrated normal inner and outer nuclear membrane after removal of BFA from the culture medium without any enlarged areas (as observed in untreated cells). In addition, the presence of distinct Golgi structure can also be seen (Fig. 6). Thus, removal of BFA resulted in a reorganization of Golgi complex and nuclear membranes. Expression of viral glycoproteins after removal of BFA
in treated
cells
We then determined whether partially glycosylated gB, gC, and gD were fully glycosylated after removal of BFA from the culture medium. In brief, HSV-1 -infected cells were treated with BFA (0.05 and 0.1 Kg per miili-
334
CHATTERJEE
AND SARKAR
FIG. 6. Electron micrograph of a BFA (0.1 pg/ml) treated human fibroblast cell after removal of the drug. Note the distinct Golgi complex (arrowheads) and nuclear membranes without any enlarged areas. Magnification, X 21,250.
liter) for 12 hr. BFA-containing medium was then replaced with growth medium without BFA. One set of treated cells, without removal of BFA, served as control. Cell lysates collected 24 hr postinfection were analyzed by immunoblot technique to determine the status of gB, gC, and gD in treated cells (i.e., continuous treatment) and in cells after removal of BFA 12 hr posttreatment. As shown in Fig. 7 the removal of BFA completely reversed the effect of this drug on the glycosylation and resulted in fully glycosylated gB, gC, and gD even after treatment for 12 hr. Restoration
of viral infectivity
after removal of BFA
Since removal of BFA reversed the effect of this drug on the glycosylation of gB, gC, and gD (probably because of reformation of a functional Golgi complex), we determined the infectivity of the intracellular HSV-1
particles after removal of BFA from the culture medium. In brief, treated and untreated cells were washed and collected in 1 ml of growth medium. Cells were sonicated for 30 set and tested for their ability to form plaques in BS-C-1 cells. Removal of BFA fully restored the infectivity of the intracellular virus particles (Table 2). As expected, removal of BFA also completely reversed the effect of this drug and restored the infectivity of extracellular HSV-1 particles (Table 3). DISCUSSION Glycoproteins are, in general, sequentially transported through the cis-, medial, and trans-compartments of the Golgi system and, finally, expressed on the cell surface (Palade, 1975; Pfeffer and Rothman, 1987; Rothman, 1981). The Golgi complex, thus, plays an important role in sorting as well as trafficking pro-
ENDOPLASMIC 6
C
123
123
A
123
RETICULUM-GOLGI
COMPLEX
CYCLING
D 123
335
PATHWAY
E
F
123
123
FIG. 7. Expression of gB, gC, and gD in BFA-treated and untreated human fibroblast cells after removal of the drug. (A, B) The strips were incubated with monoclonal antibodies to gB. (A) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (B) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. See text for details. (C. D) The strips were incubated with monoclonal antibodies to gC. (C) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (D) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. (E, F) The strips were incubated with monoclonal antibodies to gD. (E) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (F) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter; lane 3, 0.1 rg of BFA per milliliter.
teins to the cell surface. In the case of HSV-1, the glycoprotein molecules may act as nucleation points for virus assembly and budding in addition to their expression on the infected cell surface with its associated various biological functions (Chatterjee et a/., 1989; Gompels and Minson, 1986; Roizman and Batterson, 1985). Thus, HSV-1 glycoproteins have a pleiotropic role. Although some progress has been made in regard to expression and transport of HSV-1 glycoproteins in infected cells, the exact sequence of this transport pathway is unclear (Compton and Courtney, 1984; Spear, 1985). The sequence of events for HSV-1 glycoTABLE 3 REVERSALOFTHEBFA EFFECTONTHEREPLICATIONOFHSV-1' Time of treatment (hd O-24
o-12
Concentration Gglml) 0 0.05 0.1 0 0.05 0.1
No. of PFU/ml 7.9 x lo7 2.0 x 10' 0 7.9 x 10' 5.5 x 10’ 8.0 x lo7
’ Human fibroblast cells were infected with strain F and then treated with different concentrations of BFA for 24 hr (continuous treatment). BFA-containing medium was removed from one set of experiments 12 hr post-treatment and replaced by normal growth medium. Supernatants from both sets were tested for their ability to form plaques in BS-C-l cells 24 hr postinfection.
protein processing and transport is complicated by the fact that HSV-1 -infected cells express at least nine glycoproteins with a broad range of molecular weights and different functions, not all of which are clearly understood. The roles of gB, gC, and gD in various cellvirus interactions are particularly important. It is known that gB is essential for viral infectivity and is involved in penetration and cell fusion (Cai et a/., 1988; Highlander et a/., 1988; Little et al., 1981; Sarmiento et a/., 1979). Glycoprotein C is a receptor for the C3b component of complement and gC may play a role in the adsorption of HSV-1 to the cell surface (Baucke and Spear, 1979; Herold et al., 1991; Seidel-Dugan et al., 1988). Likewise, gD also plays a role in virus adsorption to the cell surface, penetration into the cells, and cell-to-cell fusion (Fuller and Spear 1985; Highlander et a/., 1987; Minson et a/., 1986; Noble et al., 1983). It has been reported that migration of VSV G protein from Golgi complex to the cell membrane was blocked in BFA-treated Chinese hamster ovary cells, leaving the G protein in a high-mannose form (Doms et a/., 1989). Recently Cheung et a/. (1991) demonstrated that HSV-1 gD contained partially processed oligosaccharides in BFA-treated cells. In this report we showed that the expression of HSV-1 gB, gC, and gD at the cell membrane were significantly blocked, probably due to their partial glycosylation at the disassembled Golgi system. Thus it is possible that partially glycosylated glycoproteins could not be transported and expressed on the surface of BFA-treated human cells. The molecular weights of all these glycoproteins were lower than
336
CHATERJEE
those of fully glycosylated, mature forms observed in untreated control. It should be noted that the glycoproteins studied were affected by BFA in, ti similar fashion in human fibroblast cells with respect to their protein profile and expression on the cell plasma membrane. As noted, in addition to being expressed on the cell surface, HSV-1 glycoproteins re also required at the nuclear membrane for assemp i”y and budding. Electron micrographs demonstrate$‘that, although restricted between the inner and out r nuclear membrane, enveloped HSV-1 particles w,d@ re formed in BFA-treated human fibroblast cells. A/similar result was reported by Whealy et al. (1991) in case of pseudorabies virus. It is interesting to note that the space between the outer and inner nuclear membrane became very enlarged in BFA-treated ccl . The exact mechanism by which BFA could induce Itf?is enlargement is unclear at present. BFA apparently did not block the normal assembly and budding of nucleocapsids as observed in the control cells. It is possible that, due to this entrapment, very few extracellular particles were present in the supernatant fluids collected from infected, BFA-treated cells. These immature, particles are not infectious as determined by plaque assay of the released HSV-1 particles from BFA-treated human fibroblast cells. In pseudorabies virus, Enquist’s group also reported that the accumulated, intracellular enveloped particles were noninfectious (Whealyetal., 1991). In any event, the trapped enveloped particles were not fully glycosylated as the Golgi complex was disassembled in the BFA-treated cells, further suggesting an important role of Golgi system in complete maturation and transport of membrane glycoproteins. Specifically, in the cis-Golgi compartment, carbohydrates like mannose are first removed. Subsequently, sialic acids, fatty acids, galactose, and others are added to the polypeptide chains in the trans-Golgi compartment (Rothman, 1981). In addition, sulfation of glycoproteins also occurs in the trans-Golgi compartment (Rothman, 1981). Thus, sequential processing of glycoproteins through the Golgi complex may be important for biological functions of the HSV-1 glycoproteins. The two events, i.e., the enlargement of the perinuclear space and the disassembly of the Golgi complex appears to be related to a single intracellular process. Interestingly, removal of BFA resulted in a redistribution and reformation of Golgi complex, leading to restoration of the infectivity of HSV-1 particles. Cheung et a/. (1991) showed that the effects of BFA on HSV propagation are not fully reversible. However, we found that removal of BFA completely restored the infectivity of HSV-1 particles. The difference observed between these two reports may be due to the type of cells employed in these studies. Recently, Chen et al. (1991) also demonstrated
AND SARKAR
that the BFA block of Punta Toro virus release is fully reversible. Alternatively, low concentrations of BFA used in our studies may also be responsible for complete restoration of the infectivity of HSV-1 after the removal of BFA. Our results indicate that there is an ER-Golgi cycling pathway inside the human fibroblast cells. The fact that BFA causes significant changes in the Golgi structure and also dilation of the ER (Doms et al., 1989) suggest that BFA might be useful for studying the intracellular processing and transport of glycoproteins in a variety of cells. In addition, BFA can provide information regarding the reformation of essential cellular organelles. ACKNOWLEDGMENTS We thank Lawrence R. Melsen and Eugene Arms of the Comprehensive Cancer Center Electron Microscope Core Facility for their excellent technical assistance and Ms. Teresa Shelton for her help in preparation of the manuscript. We also thank Richard J. Whitley for critical reviews of the manuscript. This work was supported by Public Health Service Grant Al-25120 from the National Institutes of Health.
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ENDOPLASMIC
RETICULUM-GOLGI
telns associated with the nuclear fraction of herpes simplex virus type l-infected cells. 1. viral. 49, 594-597. DOMS, R. W., Russ, G., and YEWDELL, J. W. (1989). Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum. J. Cell Biol. 109, 61-72. EBERLE, R., and COURTNEY, R. J. (1980). gA and gB glycoproteins of herpes simplex virus type 1: Two forms of a single polypeptide. J. Viral. 36, 665-675. FULLER, A. O., and SPEAR, P. G. (1985). Specificities of monoclonal and polyclonal antibodies that inhibit adsorption of herpes simplex virus to cells and lack of inhibition by potent neutralizing antibodies. 1. Virol. 55, 475-482. GLORIOSO.J.. SZCZESIUL,M. S., MARLIN, S. D., and LEVINE, M. (1983). Inhibition of glycosylation of herpes simplex virus glycoproteins: Identification of antigenic and immunogenic partially glycosylated glycopeptides on the cell surface membrane. Virology 126, l-l 8. GOMPELS, U., and MINSON, A. (1986). The properties and sequence of glycoprotein H of herpes simplex virus type 1. Virology 153, 230-247. GREENWOOD, F. C., HUNTER, W. M., and GLOVER, J. S. (1963). The preparation of I-131-labelled human growth hormone of high specific radioactivity. /. Biochem. 89, 1 14-l 23. HEROLD, B. C., WUDUNN, D., SOLP/S, N., and SPEAR, P. G. (1991). Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. /. Viral. 65, 1090-l 098. HIGHLANDER, S. L., CAI, W., PERSON, S., LEVINE, M., and GLORIOSO, J. C. (1988). Monoclonal antibodies define a domain on herpes simplex virus glycoprotein B involved in virus penetration. /. Viral. 62, 1881-1888. HIGHLANDER, S. L., SUTHERLAND, S. L., GAGE, P. J., JOHNSON, D. C.. LEVINE, M., and GLORIOSO, J. C. (1987). Neutralizing monoclonal antibodies specific for herpes simplex virus glycoprotein D inhibit virus penetration. /. Viral. 61, 3356-3364. HUTCHINSON, L., GOLDSMITH, K., PRIMORAC, S., ROOP, C., GRAHAM, F. L., and JOHNSON, D. C. (1991). Characterization of two novel HSV-1 glycoproteins, gK and gL, involved in membrane fusion. Int. Herpesvirus Workshop, 43. JOHNSON, D. A., GAUTSCH. J. W., SPORTSMAN, 1. R., and ELDER, J. H. (1984). Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose. Gene Anal. Technol. 1, 3-8. JOHNSON, D. C., and SPEAR, P. G. (1982). Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface and egress of virions from infected cells. /. Viral. 43,1102-1112. JOHNSON, D. C., and SPEAR, P. G. (1983). O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus. Cell 32, 987-997. KOGA,J., CHA~ERJEE, S., and WHITLEY, R. J. (1986). Studies on herpes simplex virus type 1 glycoproteins using monoclonal antibodies. Virology 151, 385-389. LIPPINCOTT-SCHWARTZ,J., DONALDSON, J. G., SCHWEIZER,A., BERGER, E. G., HAURI, H., YUAN, L. C., and KLAUSNER,R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggest an ER recycling pathway. Ce//60, 821-836. LITTLE, S. P.. JOFRE,1. T., COURTNEY,R. J., and SCHAFFER,P. A. (1981). A virion associated glycoprotein essential for infectivity of Herpes Simplex Virus Type 1. Virology 115, 149-l 60. LONGNECKER, R.. CHATTERJEE, S.. WHITLEY, R. J., and ROIZMAN, B.
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(1987). Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture. Proc. Nat/. Acad. Sci. USA 84, 4303-4307. MARSDEN, H. S., BUCKMASTER,A., PALFREYMAN,J. W.. HOPE, R. G., and MINSON, A. C. (1984). Characterization of the 92,000 dalton glycoprotein induced by herpes simplex virus type 2. J. Viral. 50, 547-554. MINSON, A. C., HODGMAN, T. C., DIGARD, P., HANCOCK, D. C., BELL, S. E., and BUCKMASTER,E. A. (1986). An analysis of the biological properties of monoclonal antibodies against glycoprotein D of herpes simplex virus and identification of amino acid substitutions that confer resistance to neutralization. J. Gen. Viral. 67, lOOl1013. MISUMI, Y.. MISUMI, Y., MIKI, K., TAKATSUKI,A., TAMURA, G., and IKEHARA,Y. (1986). Novel blockade by brefeldin Aof intracellulartransport of secretory proteins in cultured rat hepatocytes. /. Biol. Chem. 261, 11398-l 1403. NOBLE, A. G., LEE, G. T.-Y., SPRAGUE, R., PARISH, M. L.. and SPEAR, P. G. (1983). Anti-gD monoclonal antibodies inhibit cell fusion induced by herpes simplex virus type 1. Virology 129, 218-224. NORRILD, B., and PEDERSEN,B. (1982). Effect of tunicamycin on the synthesis of herpes simplex virus type 1 glycoproteins and their expression on the cell surface. J. Viral. 43, 395-402. PALADE, G. E. (1975). Intracellular aspects of the process of protein synthesis. Science 189, 347-358. PAUL, J. (1970). “Cell and Tissue Culture.” Churchill Livingstone, Edinburgh. PFEFFER,S. R., and ROTHMAN, J. E. (1987). Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu. Rev. Biochem. 56, 829-852. ROIZMAN, B., and BAT~ERSON,W. (1985). Herpesviruses and their replication. In “Virology” (B. N. Fields, Eds.), pp. 497-526. Raven Press, New York. ROIZMAN, B., NORRILD, B., CHAN, C., and PEREIRA.L. (1984). Identification and preliminary mapping with monoclonal antibodies of a herpes simplex virus 2 glycoprotein lacking a known type 1 counterpart. Virology 133, 242-247. ROTHMAN, J. E. (1981). The Golgi apparatus: Two organelles in tandem. Science 213, 1212-1219. SARMIENTO, M., HAFFEY, M., and SPEAR, P. G. (1979). Membrane proteins specified by herpes simplex virus. Ill. Role of glycoprotein VP7 (82) in virion infectivity. J. Viral. 29, 1 149-l 158. SEIDEL-DUGAN, C., PONCE DE LEON, M., FRIEDMAN, H. M., FRIES, L. F., FRANK, M. M.. COHEN, G. H., and EISENBERG,R. 1. (1988). C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus types 1 and 2. J. Viral. 62, 4027-4036. SPEAR, P. G. (1985). Glycoproteins specified by herpes simplex viruses. In “The Herpesviruses” (B. Roizman, Ed.), pp. 315-356. Plenum, New York. TAKATSUKI,A., and TAMUR~, G. (1985). Brefeldin A, a specific inhibitor of intracellular translocation of vesicular stomatitis virus G protein: Intracellular accumulation of high mannose type G protein and Inhibition of its cell surface expression. Agric. Biol. Chem. 49, 899-902. TOWBIN, H., STAEHELIN, T., and GORDON, 1. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nat/. Acad. Sci. USA 76,4350-4354. WHEALY, M. E., CARD, J. P., MEADE, R. P.. ROBBINS, A. K., and ENQUIST, L. W. (1991). Effect of Brefeldin A on alphaherpes virus membrane protein glycosylation and virus egress. /. Viral. 65, 1066-1081.
191,327-337
Studies
(1992)
Complex Cycling Pathway on Endoplasmic Reticulum -Golgi Virus-Infected and Brefeldin A-Treated Human Fibroblast
in Herpes Cells’
Simplex
SUBHENDRA CHAlTERJEE2 AND SAMITA SARKAR Department
of Pediatrics,
BHSB 996, University
of Alabama at Birmingham,
Birmingham,
Alabama 35294
Received May 6, 1992; accepted July 29, 1992 Brefeldin A (BFA), a fungal metabolite, significantly inhibited the release of herpes simplex virus type 1 (HSV-1) from infected human fibroblast cells. Electron micrographs of HSV-l-infected and BFA-treated human cells demonstrated the presence of enveloped particles trapped between outer and inner nuclear membranes. Analyses of viral glycoproteins B, C, and D (gB, gC, and gD) showed faster migrating, immature forms in BFA-treated cells when compared to the mature glycoproteins, as observed in the untreated control cells. The shift in mobilities of the glycoproteins in BFAtreated cells apparently was due to the disassembly of the Golgi complexwhen evaluated by an indirect immunofluorescence assay. The immature forms of gB, gC, and gD could not be detected on the surface of BFA-treated human fibroblast cells. Removal of BFA resulted in a reorganization of the Golgi complex and formation of fully glycosylated gB, gC, and gD. Moreover, the HSV-1 particles released from the treated cells after the removal of BFA completely restored the infectivity of the viral particles. Our results indicate that human fibroblast cells have an endoplasmic o 1992 Academic press, hc reticulum-Golgi cycling pathway.
perinuclear space (Spear, 1985). The virion glycoproteins are processed through the Golgi complex and converted from the “high-mannose type” (i.e., immature form) to the “complex type” (i.e., mature form). Recent studies demonstrated that brefeldin A (BFA), a fungal metabolite, blocked the transport of vesicular stomatitis virus (VSV) G protein from the ER to the Golgi complex (Doms et a/., 1989; Misumi et al., 1986; Takatsuki and Tamura, 1985). In Punta Toro virus, Chen et a/. (1991) reported that both the Gl and G2 proteins were transported out of the ER and retained in the Golgi complex. In pseudorabies virus, Enquist’s group demonstrated that although BFA had little effect on initial synthesis and modification of viral glycoproteins in the ER, it disrupted subsequent glycoprotein maturation and export (Whealy et al., 1991). Although the exact mechanism of action is unknown, it has been reported that BFA causes intracellular redistribution of the Golgi complex, leaving no identifiable structure (Doms et al., 1989; Lippincott-Schwartz et al., 1990). It has also been reported that BFA prevents the transport of HSV-1 particles from the perinuclear space into the extracellular media and the effects of BFAon HSV propagation are not fully reversible (Cheung et al., 1991). In this report, we demonstrate that BFA treatment (as little as 0.05 pg per milliliter) led to the synthesis of partially glycosylated gB, gC, and gD, probably due to the disassembly of Golgi complex. These partially glycosylated, immature forms of gB, gC, and gD were not detectable on the surface of BFA-treated, HSV-1 -infected cells. Electron micrographs of HSV-1 -infected, BFA-
INTRODUCTION Membrane glycoproteins of enveloped animal viruses are, in general, synthesized, processed, and transported inside infected cells in a sequential manner. While the intracellular transport and expression of viral glycoproteins on the cell surface are essential for many biological functions, the details of this transport pathway are poorly understood. In herpes simplex virus type 1 (HSV-1) infected cells, at least nine glycoproteins, gB, gC, gD, gE, gG, gH, gl, gK, and gL (Baucke and Spear, 1979; Eberle and Courtney, 1980; Gompels and Minson, 1986; Hutchinson et a/., 1991; Longnecker et al., 1987; Marsden et a/., 1984; Roizman et a/., 1984) have been identified. The synthesis and transport of HSV-1 glycoproteins have been studied by several investigators (Chatterjee and Burns, 1990; Chatterjee et al., 1990; Glorioso et al., 1983; Johnson and Spear, 1982; Johnson and Spear, 1983; Norrild and Pedersen, 1982) but the exact pathway for processing and transport of these glycoproteins is unclear. Viral glycoproteins, which are synthesized in the endoplasmic reticulum (ER) are transported to the nuclear membrane in an endo Hsensitive form and assembled into the virions (Compton and Courtney, 1984). These viruses bud into the
’ A portion of this investigation was presented at the General Meeting of the American Society for Microbiology, May 5-9, 1991, in Dallas, Texas. ’ To whom reprint requests should be addressed. 327
0042-6822/92
$5.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction I” any form reserved
CHAlTERJEE
328
treated cells demonstrated the presence of enveloped particles between the outer and inner nuclear membranes. The results indicate that BFA blocked intracellular processing of HSV-1 glycoproteins and migration of viral particles from the nuclear membranes to the cell surface. However, removal of BFAfrom the culture medium resulted in a reorganization and redistribution of Golgi complex and fully glycosylated gB, gC, and gD. Furthermore, the HSV-1 particles released from the treated cells following the removal of BFA completely restored the infectivity in tissue culture cells. The data indicate that BFA might be useful for studying the intracellular trafficking of glycoproteins and may provide information regarding the reformation and redistribution of cellular organelles. MATERIALS
AND METHODS
Cells and viruses African green monkey kidney (BS-C-1) cells were obtained from the American Type Culture Collection, Rockville, MD, and grown in medium 199 supplemented with 10% heat-inactivated fetal calf serum and gentamicin (50 pg/mI). Human foreskin (Hfs) fibroblast cells were prepared according to published procedures (Paul, 1970). These cells were grown in Dulbecco’s modified Eagle’s medium also supplemented with 10% heat-inactivated fetal calf serum and gentamicin (50 pglml). The F strain of HSV-1 was provided by B. Roizman, The University of Chicago, Illinois. Reagents
and radioisotopes
Brefeldin A was purchased from Epicentre Technologies (Madison, WI). Reagents for polyacrylamide gel electrophoresis were obtained from Bio-Rad Laboratories (Richmond, CA). Nalz51 (13 mCi/pg of iodine) was purchased from Amersham Corp. (Arlington Heights, IL). Protein A from Staphylococcus aureus was purchased from Pharmacia Laboratories (Uppsala, Sweden). Monoclonal antibodies against gB, gC, and gD were prepared in this laboratory (Koga et al., 1986). Rabbit anti-mouse IgG was obtained from Organon Teknika Corp. (West Chester, PA). Fluorescein-conjugated mouse IgG was purchased from Hyclone Laboratories, Inc. (Logan, UT). Rhodamine-conjugated wheat germ agglutinin was purchased from E-Y Laboratories, Inc. (San Mateo, CA). Electron
microscopy
In brief, HSV-l-infected BS-C-1 cells, grown in 60mm-diameter dishes, were treated with different concentrations of BFA. One set of cells served as untreated control. Samples were then carefully washed
AND SARKAR
with phosphate-buffered saline (PBS) 24 hr postinfection and fixed with 1% glutaraldehyde. Fixed cells were postfixed with 1% osmium tetroxide and processed for electron microscopy, as described previously (Chatterjee et al., 1982). Polyacrylamide immunoblotting
gel electrophoresis
and
Untreated and BFA-treated cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9.5%) after infection with HSV-1 (Chatterjee et a/., 1982, 1985). The fractionated proteins were electrophoretically transferred to nitrocellulose paper and processed for immunoblotting (Chatterjee et a/., 1985; Johnson et al., 1984; Towbin et a/., 1979). Nitrocellulose strips were further incubated at room temperature for 30 min with rabbit anti-mouse IgG. After immune reaction, the nitrocellulose strips were treated with [‘251]protein A for 1 hr at room temperature and then processed by autoradiography. Protein A iodination was performed as described by Greenwood et al. (1963). 1251-labeled protein A binding
immunoassay
The procedure for the protein A binding immunoassay was published previously (Chatterjee et a/., 1981, 1989). In brief, human fibroblast cells, seeded onto a 96-well microtiter plate, were treated with different concentrations of BFA after infection with HSV-1. One set of cells served as a control. The monolayers of treated and untreated cells were washed thoroughly with PBS and treated with PBS containing 0.1% bovine serum albumin (PBS-BSA) for 30 min at room temperature. After incubation with PBS-BSA, antibodies to gB, gC, or gD were added to the cells for 1 hr at room temperature. The cells were washed with PBS-BSA buffer and 5 X 1O4 cpm (approximately) of ‘251-labeled protein A was added to each well for 1 hr at room temperature. Finally, the wells were washed with PBSBSA and air dried. The viral antigen-positive wells were detected by autoradiography, as described before (Chatterjee et a/., 1981, 1989). Indirect
immunofluorescence
assay
An indirect immunofluorescence assay of virus-infected, untreated, and virus-infected, BFA-treated cells was performed in four-chamber tissue culture slides as described (Chatterjee and Burns, 1990; Chatterjee and Hunter, 1979). In brief, cells were washed with PBS and fixed in a mixture of 95% ethyl alcohol and 5% glacial acetic acid at -20” for 1 hr. The samples were treated with PBS-BSA for 30 min and then incubated with antibodies to gB or gC or gD for 1 hr at room
ENDOPLASMIC
RETICULUM-GOLGI
TABLE 1
0 0.01 0.05 0.1 0.5
No. of PFU/ml 1.0 2.0 1.0 2.0 1.0
x x x x x
106 lo4 lo2 10' 10'
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PATHWAY
Effect of BFA on the assembly HSV-1 nucleocapsids
EFFECTOF BFA ONTHERELEASEOFINFECTIOUS HSV-1 FROM HUMAN FIBROBLASTCELLS' Concentration (rdml)
COMPLEX
% Inhibition 0 98.0 99.9 99.9 99.9
a Human fibroblast cells were infected with strain F of HSV-1 and then treated with different concentrations of BFA for 18 hr. One set of cells served as untreated control. Supernatants from treated and untreated cells were then tested for their ability to form plaques in BS-C-1 cells.
temperature. Cells were washed three times with PBS-BSA and stained with fluorescein-conjugated mouse IgG. After incubation for an additional 1 hr at room temperature, cells were washed thoroughly with PBS-BSA. Finally, cells were treated with rhodamineconjugated wheat germ agglutinin for 30 min at room temperature in order to stain the Golgi complex. Cells were examined by episcopic fluorescence using a Nikon Fluorophot microscope.
RESULTS
329
and budding
of
The intracellular status of HSV-1 nucleocapsids was determined with regard to assembly and budding in BFA-treated cells. Untreated and BFA-treated human fibroblast cells were processed for electron microscopy after HSV-1 infection. Electron micrographs demonstrated HSV-1 nucleocapsids inside the nuclei of both untreated and BFA-treated cells even at a concentration of 0.1 fig/ml (Fig. 1). In addition to the nucleocapsids, enveloped HSV-1 particles were also seen in BFA-treated cells but were restricted to the space between outer and inner nuclear membrane (Fig. 1B, 1C). It was noticed that the space between the outer and inner nuclear membrane became enlarged in BFA-treated cells. No distinct Golgi structure was observed inside the BFA-treated ceils when compared to that observed with the untreated cells (Fig. 1A). Intracellular viral infectivity of the BFA-treated cells was significantly reduced as determined by a plaque assay. In brief, human fibroblast cells were infected and treated with 0.05 and 0.1 pg/ml of BFA for 24 hr. Treated and untreated cells were washed twice and collected in 1 ml of growth medium. Cells were sonicated for 30 set and tested for their ability to form plaques in BS-C-l cells. The result of this experiment showed a significant reduction in the synthesis of infectious particles inside the BFA-treated (0.05 pg/ml) cells (Table 2, upper panel).
Effect of BFA on the release of HSV-1 from human fibroblast cells To determine the dose-response effects of BFA, human fibroblast cells were infected with F strain of HSV1 (multiplicity of infection = 5) for 1 hr and then treated with 0.01, 0.05, 0.1, and 0.5 pg/ml of BFA. One set of cells served as untreated control. Supernatant fluids from treated and untreated cells were collected 18 hr postinfection and tested for their ability to form plaques in BS-C-1 cells. Even 0.01 pglml of BFA significantly blocked the release of infectious HSV-1 from human fibroblast cells (Table 1). These results were confirmed by i,mmuno dot blot evaluation of extracellular viral particles. In brief, culture supernatants collected from BFA-treated and untreated cells 18 hr postinfection were clarified and the virus was pelleted by centrifugation at 40,000 rpm for 1 hr. The pellets were processed for a dot blot assay using rabbit antiserum to HSV-1 in order to determine the total quantity of extracellular viral particles released from treated and untreated cells. A reduction of 97.25% in the quantity of total extracellular viral particles released from BFA-treated (0.05 pg/ml) cells was noticed.
Expression
of viral glycoproteins
in BFA-treated
cells
Since enveloped viral particles were observed within BFA-treated cells, the status of the selected glycoproteins in treated cells was determined. Control and BFA-treated (0.05 and 0.1 pg/ml) cell lysates collected 18 hr postinfection were analyzed by immunoblot technique to assess the expression of gB, gC, or gD using monoclonal antibodies. As displayed in Fig. 2, gB, gC, and gD from BFA-treated cells migrated faster than that observed in the untreated control cells. Thus, the glycoproteins expressed in the presence of BFA appeared only partially glycosylated. In parallel, we also examined the incorporation of glycoproteins into the extracellular viral particles (approximately 3.4 and 2%) released from BFA-treated (0.05 and 0.1 pg per milliliter, respectively) cells. Figure 3 shows that the expression of HSV-1 glycoproteins on the extracellular virus particles produced from BFA-treated cells was completely inhibited. The quantities of extracellular virus particles released from BFA-treated cells were determined by incubating the same nitrocellulose blot
330
CHAlTERJEE
AND SARKAR
FIG. 1. Electron micrographs of HSV-1 particles in BFA-treated and untreated human fibroblast cells. (A) Untreated cell. Distinct inner and outer nuclear membranes and Golgi complex (G) can be observed. Extracellular and intracellular virus particles(V) can also be seen. Magrt ification, X 53,600. (B) Cells treated with BFA (0.1 pg/ml) showing virus particles trapped between the outer and inner nuclear membrane (ar‘rowheads). No distinct Golgi complex was observed. Magnification, X 19,680. (C) Magnified view of a BFA (0.1 pglml) treated cell. Note the enlar -ged perinuclear area with several enveloped virus particles trapped inside. Magnification, X 88,000.
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FIG. 1 -Continued
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332
CHATTERJEE AND SARKAR
A
TABLE 2 INFECTIVITYOF THE INTRACELLULARVIRUS PARTICLESIN THE CONTINUOUS PRESENCEOF BFA AND AFTERTHE BFA IS REMOVED Time of treatment 0-d In presence O-24
of BFA
After removal of BFA o-12
with rabbit antiserum counting.
123
No. of PFU/ml
Concentration 0 0.05 0.1
7.0 x lo8 3.0 x 10’ 2.0 x 10’
0 0.05 0.1
7.0 x lo8 7.1 x 106 6.9 x lo6
to HSV-1 followed
Localization of HSV-1 glycoproteins human fibroblast cells
123
B
by gamma
in BFA-treated
Since gB, gC, and gD were expressed in the BFAtreated cells, albeit improperly, we localized these glycoproteins within the BFA-treated cells in order to define the block in glycosylation. Control and BFA-treated (0.1 pg/ml), HSV-1 -infected cells were examined by indirect immunofluorescence as shown in Fig. 4. The result of this experiment demonstrated that BFA caused disassembly of the Golgi complex, leaving no definable structure as evident from the staining pattern with rhodamine-conjugated wheat germ agglutinin
A
B
C
123
123
123
FIG. 3. Cells were infected with strain F and then treated with different concentrations of BFA for 18 hr. One set of cells served as untreated control. Culture supernatants were collected, clarified, and the virus was pelleted by centrifugation at 40,000 rpm for 1 hr. The pellets werefysed and equal amounts (50 ~1)from untreated and treated samples were subjected to polyacrylamide gel electrophoresis and immunoblotting. The nitrocellulose blot was reacted with monoclonal antibodies to gB, gC. and gD. (A) The strip was incubated with monoclonal antibodies to gB and gD. (B) The strip was incubated with monoclonal antibodies to gC. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter: lane 3. 0.1 pg of BFA per milliliter.
(Fig. 4C). In contrast, a distinct Golgi complex could be observed in untreated control cells (Fig. 4A). It is possible that this defect in Golgi complex was responsible for altered or diffused patterns and reduced expression of gC in BFA-treated human cells as evident from the fluorescein-labeled cells (Fig. 4D). Although some cells apparently displayed normal staining patterns, the glycoproteins in these cells were also partially glycosylated as no mature glycoproteins were observed in the immunoblot profiles. A similar pattern of immunofluorescence was observed when antibodies against gB and gD were used instead of antibodies to gC (data not shown). Expression of HSV-1 glycoproteins on the surface of BFA-treated human fibroblast cells
FIG. 2. Expression of HSV-1 gB, gC, and gD in BFA-treated and untreated human fibroblast cells. (A) The strip was incubated with monoclonal antibody to gB. (B) The strip was incubated with monoclonal antibody to gC. (C) The strip was incubated with monoclonal antibody to gD. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter; lane 3, 0.1 rg of BFA per milliliter.
Although expression of gB, gC, and gD was observed in BFA-treated cells, it was not possible from the above experiments to determine whether these glycoproteins were expressed on the surface of BFAtreated cells. To detect the presence of gB, gC, and gD on the surface of BFA-treated cells, ‘251-labeled protein A binding immunoassay was performed. As shown in Fig. 5, 0.1 pg/ml of BFA significantly inhibited the ex-
ENDOPIASMIC
RETICULUM-GOLGI
COMPLEX
CYCLING
333
PATHWAY
FIG. 4. Indirect immunofluorescence assay of BFA-treated and untreated human cells after HSV-1 infection. All samples were reacted with monoclonal antibody to gC. (A) Untreated fluorescein-labeled cells reacted with rhodamine-conjugated wheat germ agglutinin. (B) Untreated cells, fluorescein labeled. The arrows indicate the positions of Golgi complexes. (C) Cells treated with BFA (0.1 pg/ml), fluorescein labeled, and reacted with rhodamine-conjugated wheat germ agglutinin. (D) Cells treated with BFA (0.1 pg/ml), fluorescein labeled. The arrows indicate the positions of disassembled Golgi complexes.
pression of gB, gC, and gD on the surface of infected cells. Structural reorganization removal of BFA
of treated
cells after
To determine whether these effects of BFA were reversible after removal of the drug from the culture me1 2 3 ABC
DE
F
GH
I
FIG. 5. Autoradiogram of 1251-labeled protein A binding assay to demonstrate the expression of HSV-1 glycoproteins on the surface of BFA-treated and untreated human fibroblast cells. Rows A, B. C, cells incubated with monoclonal antibody to gC. Rows D, E, F. cells incubated with monoclonal antibody to gD. Rows G, H, I, cells incubated with monoclonal antibody to gB. Rows, A, D, G, untreated cells. Rows B, E, H, cells treated with 0.1 rg of BFA per milliliter. Rows C, F, I, cells treated with 0.5 fig/ml of BFA per milliliter. Wells in row 1, uninfected control cells: wells in row 2, cells infected with 2 PFU/cell; wells in row 3, cells infected with 0.5 PFU/cell.
dia, HSV-1 -infected human fibroblast cells were treated with BFA at a concentration of 0.05 or 0.1 pg per milliliter. Twelve hour postinfection BFA containing medium was replaced by growth medium without BFA. Ceils were then processed for electron microscopy 24 hr postinfection as described under Materials and Methods. The resulting electron micrograph (Fig. 6) demonstrated normal inner and outer nuclear membrane after removal of BFA from the culture medium without any enlarged areas (as observed in untreated cells). In addition, the presence of distinct Golgi structure can also be seen (Fig. 6). Thus, removal of BFA resulted in a reorganization of Golgi complex and nuclear membranes. Expression of viral glycoproteins after removal of BFA
in treated
cells
We then determined whether partially glycosylated gB, gC, and gD were fully glycosylated after removal of BFA from the culture medium. In brief, HSV-1 -infected cells were treated with BFA (0.05 and 0.1 Kg per miili-
334
CHATTERJEE
AND SARKAR
FIG. 6. Electron micrograph of a BFA (0.1 pg/ml) treated human fibroblast cell after removal of the drug. Note the distinct Golgi complex (arrowheads) and nuclear membranes without any enlarged areas. Magnification, X 21,250.
liter) for 12 hr. BFA-containing medium was then replaced with growth medium without BFA. One set of treated cells, without removal of BFA, served as control. Cell lysates collected 24 hr postinfection were analyzed by immunoblot technique to determine the status of gB, gC, and gD in treated cells (i.e., continuous treatment) and in cells after removal of BFA 12 hr posttreatment. As shown in Fig. 7 the removal of BFA completely reversed the effect of this drug on the glycosylation and resulted in fully glycosylated gB, gC, and gD even after treatment for 12 hr. Restoration
of viral infectivity
after removal of BFA
Since removal of BFA reversed the effect of this drug on the glycosylation of gB, gC, and gD (probably because of reformation of a functional Golgi complex), we determined the infectivity of the intracellular HSV-1
particles after removal of BFA from the culture medium. In brief, treated and untreated cells were washed and collected in 1 ml of growth medium. Cells were sonicated for 30 set and tested for their ability to form plaques in BS-C-1 cells. Removal of BFA fully restored the infectivity of the intracellular virus particles (Table 2). As expected, removal of BFA also completely reversed the effect of this drug and restored the infectivity of extracellular HSV-1 particles (Table 3). DISCUSSION Glycoproteins are, in general, sequentially transported through the cis-, medial, and trans-compartments of the Golgi system and, finally, expressed on the cell surface (Palade, 1975; Pfeffer and Rothman, 1987; Rothman, 1981). The Golgi complex, thus, plays an important role in sorting as well as trafficking pro-
ENDOPLASMIC 6
C
123
123
A
123
RETICULUM-GOLGI
COMPLEX
CYCLING
D 123
335
PATHWAY
E
F
123
123
FIG. 7. Expression of gB, gC, and gD in BFA-treated and untreated human fibroblast cells after removal of the drug. (A, B) The strips were incubated with monoclonal antibodies to gB. (A) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (B) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. See text for details. (C. D) The strips were incubated with monoclonal antibodies to gC. (C) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (D) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. (E, F) The strips were incubated with monoclonal antibodies to gD. (E) Cell lysates from treated cells without removal of BFA (i.e., continuous treatment). (F) Cell lysates from treated cells after removal of BFA 12 hr post-treatment. Lane 1, No BFA; lane 2, 0.05 pg of BFA per milliliter; lane 3, 0.1 rg of BFA per milliliter.
teins to the cell surface. In the case of HSV-1, the glycoprotein molecules may act as nucleation points for virus assembly and budding in addition to their expression on the infected cell surface with its associated various biological functions (Chatterjee et a/., 1989; Gompels and Minson, 1986; Roizman and Batterson, 1985). Thus, HSV-1 glycoproteins have a pleiotropic role. Although some progress has been made in regard to expression and transport of HSV-1 glycoproteins in infected cells, the exact sequence of this transport pathway is unclear (Compton and Courtney, 1984; Spear, 1985). The sequence of events for HSV-1 glycoTABLE 3 REVERSALOFTHEBFA EFFECTONTHEREPLICATIONOFHSV-1' Time of treatment (hd O-24
o-12
Concentration Gglml) 0 0.05 0.1 0 0.05 0.1
No. of PFU/ml 7.9 x lo7 2.0 x 10' 0 7.9 x 10' 5.5 x 10’ 8.0 x lo7
’ Human fibroblast cells were infected with strain F and then treated with different concentrations of BFA for 24 hr (continuous treatment). BFA-containing medium was removed from one set of experiments 12 hr post-treatment and replaced by normal growth medium. Supernatants from both sets were tested for their ability to form plaques in BS-C-l cells 24 hr postinfection.
protein processing and transport is complicated by the fact that HSV-1 -infected cells express at least nine glycoproteins with a broad range of molecular weights and different functions, not all of which are clearly understood. The roles of gB, gC, and gD in various cellvirus interactions are particularly important. It is known that gB is essential for viral infectivity and is involved in penetration and cell fusion (Cai et a/., 1988; Highlander et a/., 1988; Little et al., 1981; Sarmiento et a/., 1979). Glycoprotein C is a receptor for the C3b component of complement and gC may play a role in the adsorption of HSV-1 to the cell surface (Baucke and Spear, 1979; Herold et al., 1991; Seidel-Dugan et al., 1988). Likewise, gD also plays a role in virus adsorption to the cell surface, penetration into the cells, and cell-to-cell fusion (Fuller and Spear 1985; Highlander et a/., 1987; Minson et a/., 1986; Noble et al., 1983). It has been reported that migration of VSV G protein from Golgi complex to the cell membrane was blocked in BFA-treated Chinese hamster ovary cells, leaving the G protein in a high-mannose form (Doms et a/., 1989). Recently Cheung et a/. (1991) demonstrated that HSV-1 gD contained partially processed oligosaccharides in BFA-treated cells. In this report we showed that the expression of HSV-1 gB, gC, and gD at the cell membrane were significantly blocked, probably due to their partial glycosylation at the disassembled Golgi system. Thus it is possible that partially glycosylated glycoproteins could not be transported and expressed on the surface of BFA-treated human cells. The molecular weights of all these glycoproteins were lower than
336
CHATERJEE
those of fully glycosylated, mature forms observed in untreated control. It should be noted that the glycoproteins studied were affected by BFA in, ti similar fashion in human fibroblast cells with respect to their protein profile and expression on the cell plasma membrane. As noted, in addition to being expressed on the cell surface, HSV-1 glycoproteins re also required at the nuclear membrane for assemp i”y and budding. Electron micrographs demonstrate$‘that, although restricted between the inner and out r nuclear membrane, enveloped HSV-1 particles w,d@ re formed in BFA-treated human fibroblast cells. A/similar result was reported by Whealy et al. (1991) in case of pseudorabies virus. It is interesting to note that the space between the outer and inner nuclear membrane became very enlarged in BFA-treated ccl . The exact mechanism by which BFA could induce Itf?is enlargement is unclear at present. BFA apparently did not block the normal assembly and budding of nucleocapsids as observed in the control cells. It is possible that, due to this entrapment, very few extracellular particles were present in the supernatant fluids collected from infected, BFA-treated cells. These immature, particles are not infectious as determined by plaque assay of the released HSV-1 particles from BFA-treated human fibroblast cells. In pseudorabies virus, Enquist’s group also reported that the accumulated, intracellular enveloped particles were noninfectious (Whealyetal., 1991). In any event, the trapped enveloped particles were not fully glycosylated as the Golgi complex was disassembled in the BFA-treated cells, further suggesting an important role of Golgi system in complete maturation and transport of membrane glycoproteins. Specifically, in the cis-Golgi compartment, carbohydrates like mannose are first removed. Subsequently, sialic acids, fatty acids, galactose, and others are added to the polypeptide chains in the trans-Golgi compartment (Rothman, 1981). In addition, sulfation of glycoproteins also occurs in the trans-Golgi compartment (Rothman, 1981). Thus, sequential processing of glycoproteins through the Golgi complex may be important for biological functions of the HSV-1 glycoproteins. The two events, i.e., the enlargement of the perinuclear space and the disassembly of the Golgi complex appears to be related to a single intracellular process. Interestingly, removal of BFA resulted in a redistribution and reformation of Golgi complex, leading to restoration of the infectivity of HSV-1 particles. Cheung et a/. (1991) showed that the effects of BFA on HSV propagation are not fully reversible. However, we found that removal of BFA completely restored the infectivity of HSV-1 particles. The difference observed between these two reports may be due to the type of cells employed in these studies. Recently, Chen et al. (1991) also demonstrated
AND SARKAR
that the BFA block of Punta Toro virus release is fully reversible. Alternatively, low concentrations of BFA used in our studies may also be responsible for complete restoration of the infectivity of HSV-1 after the removal of BFA. Our results indicate that there is an ER-Golgi cycling pathway inside the human fibroblast cells. The fact that BFA causes significant changes in the Golgi structure and also dilation of the ER (Doms et al., 1989) suggest that BFA might be useful for studying the intracellular processing and transport of glycoproteins in a variety of cells. In addition, BFA can provide information regarding the reformation of essential cellular organelles. ACKNOWLEDGMENTS We thank Lawrence R. Melsen and Eugene Arms of the Comprehensive Cancer Center Electron Microscope Core Facility for their excellent technical assistance and Ms. Teresa Shelton for her help in preparation of the manuscript. We also thank Richard J. Whitley for critical reviews of the manuscript. This work was supported by Public Health Service Grant Al-25120 from the National Institutes of Health.
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