Department of Molecular, Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309, USA
Correspondence
Jennifer Martin
jm{at}Colorado.edu
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ABSTRACT |
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INTRODUCTION |
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A small percentage of cells within most EBV-positive human lymphoblastoid cell lines support lytic infection. The EBV-positive B lymphoblastoid cell line B95-8, derived from infected marmoset lymphocytes, produces more virus than human lymphoblastoid cell lines and the efficiency of virus production can be increased by treatment with chemical inducers such as phorbol esters (Miller, 1989; Miller & Lipman, 1973
). Herpesvirus capsids, assembled in the nucleus, bud through the inner nuclear membrane and thereby acquire tegument and a primary envelope (Gong & Kieff, 1990
; Steven & Spear, 1997
). How the maturing virus particle moves from the perinuclear space to the plasma membrane is not well understood. The primary envelope can fuse with the outer nuclear membrane, resulting in nucleocapsid release into the cytoplasm (Gong & Kieff, 1990
). EBV has also been proposed to leave the perinuclear space via budding through the outer nuclear membrane in vesicles, which then release nucleocapsids into the cytoplasm (Gong & Kieff, 1990
; Torrisi et al., 1989
). The virus may acquire its final envelope either by budding through the plasma membrane or by budding into post-Golgi-derived vesicles followed by exocytosis of the enveloped virion (Gong & Kieff, 1990
). Thus, a process of nucleocapsid envelopment, de-envelopment and re-envelopment must occur during EBV egress.
EBV encodes two related membrane proteins, latent membrane protein-1 (LMP-1) and lytic LMP-1 (lyLMP-1). The two proteins are differentially expressed during the virus life cycle in vitro (Hudson et al., 1985). LMP-1 is expressed during latent infection and is essential for human B cell immortalization by EBV (Fennewald et al., 1984
; Kaye et al., 1993
). LMP-1's constitutive tumour necrosis factor receptor-associated factor (TRAF)-dependent activity contributes both proliferative and survival signals to the immortalized cell (Eliopoulos & Rickinson, 1998
; Izumi et al., 1997
; Kilger et al., 1998
; Zimber-Strobl et al., 1996
). LMP-1 is a multispanning membrane protein and accumulates at the surface of the infected cell and in intracellular membranes (Mann et al., 1985
). Immunofluorescent analysis of the subcellular localization of LMP-1 in a variety of cell types, including B cells, reveals patched staining at the surface and within intracellular membrane compartments (Liebowitz et al., 1986
; Mann et al., 1985
). LMP-1 may signal, possibly as an oligomer, from lipid microdomains (lipid rafts) in the plasma membrane (Higuchi et al., 2001
; Kaykas et al., 2001
).
LyLMP-1 is categorized as a late lytic-cycle protein in permissive B95-8 cells (Hudson et al., 1985). LyLMP-1 encodes the C-terminal 258 aa of LMP-1 and thus is comprised of the fifth and sixth transmembrane domains and the C terminus of LMP-1. Although lyLMP-1 retains the intracellular signalling domain of LMP-1, it does not bind TRAFs nor does it activate TRAF-dependent signalling pathways (Baichwal & Sugden, 1989
; Erickson & Martin, 2000
; Huen et al., 1995
; Mitchell & Sugden, 1995
; Wang et al., 1988a
, b
). LyLMP-1 can inhibit LMP-1 signalling to NF-
B and JNK when the two proteins are coexpressed at levels mimicking those observed in phorbol 12-tetradecanoate 13-acetate (TPA) and butyrate (TPA/butyrate)-induced B95-8 cells. Inhibition does not result from the sequestration of lyLMP-1 of critical TRAF proteins nor from disruption by LMP-1 oligomers (Erickson & Martin, 1997
, 2000
). Immunofluorescent studies of infected B cells show that, unlike LMP-1, lyLMP-1 staining is not patched in cell membranes but is diffuse and localized to intracellular membranes (Wang et al., 1988a
, b
). LyLMP-1 immunoreactivity has been localized to cell-free, extracellular EBV virions in B95-8 cell supernatants (Erickson & Martin, 1997
).
More precise localization of LMP-1 proteins in EBV-infected B cells was undertaken to reveal clues to their function in the EBV life cycle in general and to models of lyLMP-1 inhibition of LMP-1 signalling. Here we report the ultrastructural localization of LMP-1 and lyLMP-1 determined by immunoelectron microscopy of rapidly frozen, latently infected, B lymphoblastoid cells and permissive B95-8 cells. LMP-1 immunoreactivity was clustered at the surface of 721 cells, in cell surface vesicles budding to the outside of the cell and in small vesicles accumulating in 721 cell culture medium. LyLMP-1 labelling was detected only in B95-8 cells actively producing virus and was concentrated in nuclear membranes early when virus particles were in the nucleus, and in cytoplasmic and plasma membranes late when virus particles accumulated in the cytoplasm. Finally, we found lyLMP-1 immunoreactivity associated with both intracellular and extracellular enveloped virions.
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METHODS |
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Lytic cycle induction.
B95-8 cells were grown to saturation (106 cells ml-1), treated with 20 ng TPA ml-1 and 3·5 mM sodium butyrate (TPA/butyrate) for 72 h and harvested by centrifugation. Cell pellets were processed for Western blot analysis or electron microscopy (EM).
Antibodies.
The primary antiserum (anti-LMP-1) used for immunochemistry was raised in rabbits against the LMP-1 C terminus fused to glutathione S-transferase and affinity-purified; anti-LMP-1 antiserum recognizes both LMP-1 and lyLMP-1. 72A1 is a mouse monoclonal antibody that recognizes the EBV lytic cycle protein gp350/220 (a gift from L. Hutt-Fletcher). Secondary antibodies include: alkaline phosphatase-conjugated anti-rabbit IgG (Sigma); FITC-conjugated anti-rabbit IgG (Sigma); horseradish peroxidase-conjugated anti-rabbit IgG (Promega); goat anti-rabbit IgG conjugated to 10 or 15 nm gold particles; and goat anti-mouse IgG/IgM conjugated to 15 nm gold particles (Ted Pella).
Western blot analysis.
Whole cell lysates and purified membrane fractions were analysed by SDS-PAGE (10 % gels) and Western blot using anti-LMP-1 antiserum, as described previously (Coffin et al., 2001; Erickson & Martin, 1997
).
Immunofluorescence analysis.
Cells were fixed on coverslips and stained for LMP-1, as described previously (Coffin et al., 2001).
High-pressure freezing.
Cell cultures of 105106 cells ml-1 were harvested by centrifugation and resuspended in a cryoprotectant solution of 150 mM mannitol in RPMI medium supplemented with 10 % calf serum. Pelleted cells were transferred into a brass specimen carrier and inserted into the BAL-TEC HPM-010 high-pressure freezer (Technotrade International) (Giddings et al., 2001). Frozen samples were transferred to liquid nitrogen and maintained at -196 °C until freeze-substitution.
Freeze-substitution and embedding in Lowicryl HM20.
High-pressure frozen samples were freeze-substituted in 0·25 % glutaraldehyde/0·1 % uranyl acetate in acetone at -80 °C for 3 days and incubated at -20 °C for 1 day. Fixed and dehydrated cells were then infiltrated with a series of acetone/Lowicryl HM20 mixtures at -20 °C until 100 % HM20 resin was attained. After several changes with fresh HM20 at -20 °C, samples were loaded into plastic embedding capsules and the resin was polymerized by exposure to UV at -50 °C (Giddings et al., 2001).
Freeze-substitution and embedding in Epon-Araldite.
Frozen cells were transferred to freeze-substitution medium composed of 0·5 % glutaraldehyde/0·05 % tannic acid in acetone. Samples were stored at -80 °C for 2 days, rinsed in acetone at -80 °C and transferred to 2 % osmium tetroxide in acetone at the same temperature. Specimens were warmed up gradually to room temperature over 24 h and then rinsed with acetone. Samples were then infiltrated with Epon resin.
Immunolabelling of freeze-substituted samples.
Sections, 5060 nm thick, were collected on Formvar-coated nickel grids. Nonspecific antibody binding was blocked by incubation in 2 % non-fat dry milk in PBS/0·1 % Tween 20 (PBST). Samples were incubated in primary antibody (1 : 20 in blocking buffer) and then rinsed with PBST. Grids were transferred to secondary antibody (1 : 5 or 1 : 20 in PBST), then rinsed in PBST followed by water. Following antibody labelling, samples were stained with 2 % uranyl acetate in either water or 70 % methanol/water and post-stained with Reynold's lead citrate. Samples were analysed at 80 kV on a Philips CM10 transmission electron microscope.
Surface immunolabelling of intracellular and extracellular virions.
B95-8 cells were treated with TPA/butyrate for 96 h and harvested by centrifugation. Cell pellets were resuspended and incubated with anti-LMP-1 antiserum (1 : 10) in blocking solution (2 % BSA/1x PBS) for 45 min on ice. Cells were resuspended in blocking solution and incubated in goat anti-rabbit secondary antibody conjugated to gold (1 : 5) in blocking solution for 45 min on ice. After washing with blocking solution, cells were fixed with Karnovsky's fixative (5 % glutaraldehyde/4 % formaldehyde) overnight, followed by fixation with 1 % osmium tetroxide. Finally, specimens were dehydrated gradually with increasing concentrations of ethanol mixtures up to 100 % ethanol, then embedded in Epon. Blocks were sectioned, stained and visualized by EM as described above.
Fractionation of 721 cell culture medium.
721 cells were washed twice in PBS, plated at 4x105 cells ml-1 in 35 ml RPMI medium supplemented with 10 % calf serum and grown for 2448 h. Differential centrifugation of 721 cell supernatants was performed essentially as described (Raposo et al., 1996
). Cells were removed by centrifugation at 300 g and the medium was centrifuged sequentially at 300 g (10 min), 1200 g (twice for 10 min), 10 000 g (30 min), 70 000 g (1 h) and 100 000 g (1 h) (10 000, 70 000 and 100 000 g spins were performed in an SW27 rotor). Pellets were resuspended in 20 ml PBS and recentrifuged at 100 000 g for 1 h. Final pellets were resuspended in 25 µl PBS. Samples were diluted 1 : 1 with 4x SDS sample buffer and one-quarter of each pellet was analysed by Western blot with anti-LMP-1 antiserum, as described above. The pellet from the 70 000 g spin was analysed by sucrose gradient centrifugation as described below.
Sucrose density centrifugation of the 70 000 g pellet from 721 cell supernatants.
Sucrose gradient centrifugation was performed as described previously (Raposo et al., 1996). Briefly, the 70 000 g pellet [from fractionated cell medium (above)] was resuspended in 2·5 M sucrose/20 mM HEPES/NaOH, pH 7·2. A linear sucrose gradient (20·25 M sucrose/20 mM HEPES/NaOH, pH 7·2) was layered on top of the resuspended pellet and centrifuged in an SW27 rotor at 100 000 g for 15 h. Fractions were taken from the top of the gradient (18x2 ml), diluted with 2 ml PBS and recentrifuged for 1 h at 200 000 g in an SW50.1 rotor. Resulting pellets were analysed by EM (see below) or SDS-PAGE. For SDS-PAGE, pellets were resuspended in 100 µl 4x SDS sample buffer and 15 µl of each fraction was analysed by SDS-PAGE, as described above. For EM analysis, pellets were resuspended in PBS and processed as described below.
Immunoelectron microscopic analysis of the 70 000 g pellet.
Pellets from sucrose gradient fractions 712 were resuspended and pooled in 30 ml PBS. Pooled material was pelleted in an SW27 rotor at 70 000 g for 1 h and the resulting pellet was resuspended in 20 µl 0·02 % sodium azide/PBS, fixed immediately with 2 % paraformaldehyde, placed on copper grids and air-dried. Grids were blocked with 1 % BSA/PBS for 30 min, incubated in anti-LMP-1 antiserum for 45 min and labelled with secondary antibody (anti-rabbit antibody conjugated with 15 nm gold) for 45 min. Grids were stained with 1 % uranyl acetate and visualized with a Philips Technai F20 transmission electron microscope operating at 80 kV.
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RESULTS |
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Identification of LMP-1 in plasma membrane protrusions
To determine if LMP-1-enriched plasma membrane protrusions represented a stage in the process of plasma membrane budding, we asked if LMP-1 protein accumulated in the supernatant of 721 cells. Conditioned media from 721 cells was fractionated by differential centrifugation and fractions were assayed by Western blot for LMP-1 (Fig. 2A). Full-length LMP-1 was found in the cell pellet (300 g spin) and 1200 g pellets containing broken cell debris. Importantly, LMP-1 was found in the pellet from the 70 000 g spin. Previous work has demonstrated that 70 000 g pellets harvested from conditioned medium from EBV-positive B lymphoblastoid cells contain small membrane vesicles of 6080 nm in diameter, called exosomes, which could activate T cells (Raposo et al., 1996
). The LMP-1 fractionating with the 70 000 g pellet may be contained in exosomes or perhaps in small membrane vesicles derived from the plasma membrane. To determine if the LMP-1 immunoreactivity in the 70 000 g pellet was membrane bound, material from the 70 000 g pellet was analysed by sucrose gradient centrifugation. LMP-1 protein floated to an equilibrium density of 1·13 g ml-1, confirming its association with membrane vesicles (Fig. 2B
). Pelleted material from fractions 712 (corresponding to densities of 1·1211·213 g ml-1) was analysed by immunoelectron microscopy to determine the size and homogeneity of LMP-1-containing vesicles. LMP-1 labelling was observed in small membrane vesicles of fairly homogeneous size, with an average diameter of
80 nm (Fig. 2C
). Thus, LMP-1-containing vesicles exhibit properties very similar to exosomes isolated from supernatants of EBV-immortalized lymphoblastoid cell lines (Escola et al., 1998
; Raposo et al., 1996
).
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Cell pellets from uninduced B95-8 cells were examined. No specific pattern of immunolabelling could be detected in uninduced B95-8 cells. Nonspecific labelling with gold particles was observed, at very low density, throughout the cell (not shown). Qualitative Western blot analysis revealed that uninduced B95-8 cells expressed significantly less full-length LMP-1 than did 721 cells on a population level (Fig. 3). Therefore, the lack of specific LMP-1 labelling in uninduced B95-8 cells resulted from the fact that full-length LMP-1 levels were below the limit of detection of our immunogold-labelling method. Localization of full-length LMP-1 in B95-8 cells was, therefore, not possible.
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In contrast to uninduced B95-8 cells, specific labelling was observed in B95-8 cells treated with TPA/butyrate for 72 h (Figs 4 and 5). Immunolabelling was observed in the nuclear membrane, plasma membrane and, to a lesser extent, in cytoplasmic membranes. The pattern of gold labelling was distinct from that observed for LMP-1 in 721 cells in that it was rarely organized in patches' or clusters in the membrane (Figs 4 and 5
). Importantly, specific labelling was observed only in cells producing virus (i.e. cells in which intracellular virus was detected by EM) (Table 1
). The lack of labelling in uninduced cells, the restriction of labelling to cells producing virus, and the lack of similarity in labelling pattern between TPA/butyrate-induced B95-8 cells and 721 cells indicated that immunogold labelling in TPA/butyrate-induced B95-8 cells reflected lyLMP-1, and not full-length LMP-1, localization.
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Morphological examination of cytoplasmic virus particles revealed that many resembled nucleocapsids found inside the nucleus. To enhance morphological preservation of the viral envelope and to determine if cytoplasmic virus particles were naked nucleocapsids, TPA/butyrate-induced B95-8 cells were fixed with osmium after high-pressure freezing and embedded in Epon resin (Fig. 6). Extracellular enveloped virions ranged in size from 100 to 150 nm in diameter, whereas nuclear and many cytoplasmic nucleocapsids were generally less than 100 nm in diameter. These observations suggest that many virus particles in the cytoplasm lacked envelopes and are consistent with a process of rapid de- and re-envelopment of the nucleocapsid during virus egress, as suggested previously (Gong & Kieff, 1990
).
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To confirm that lyLMP-1 was virion-associated in cell samples used for immunogold labelling (above), released virus in cell-free supernatants was labelled and stained as described previously (Erickson & Martin, 1997). Negative staining confirmed our previous findings that lyLMP-1 is associated with virions in TPA/butyrate-induced B95-8 supernatants (not shown; Erickson & Martin, 1997
). To facilitate antibody access to antigen, the cell sample was embedded after antibody incubation (surface labelling). Intact TPA/butyrate-induced B95-8 cells were incubated with anti-LMP-1 antiserum, fixed with osmium, embedded in Epon and processed for EM analysis (see Methods). Data shown in Fig. 7
are from B95-8 cells treated with TPA/butyrate for 96 h. These cells are actively producing virus and are quite fragile and leaky. Therefore, the primary antisera presumably gained access to intracellular sites by leakage. This approach clearly revealed lyLMP-1 labelling of intracellular and extracellular virions and suggested that our failure to visualize labelled intracellular virions using high-pressure freezing and immunolabelling was due in part to problems with sample preparation (Fig. 7A, B
). These results suggest lyLMP-1 may be incorporated into cytoplasmic virions when budding through the nuclear membrane and into extracellular virions when budding through the plasma membrane.
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DISCUSSION |
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LMP-1 localization in latently infected lymphoblastoid cells
LMP-1, not surprisingly, was localized primarily in the plasma membrane of latently infected lymphoblastoid cells (Fig. 1B). LMP-1 localization in clusters at the plasma membrane is consistent with its lipid raft localization. However, LMP-1 labelling in plasma membrane protrusions' (Fig. 1C
) and in 80 nm vesicles in conditioned medium (Fig. 2C
) was unexpected. The size of LMP-1-positive vesicles is consistent with LMP-1 being shed into the extracellular space via exosomes or microvesicles. Interestingly, Dukers et al. (2000)
reported, but did not characterize, LMP-1 immunoreactivity in conditioned medium from EBV-positive lymphoblastoid cells. LMP-1 turns over rapidly in 721 cells with a half-life of 2 h (Baichwal & Sugden, 1987
). LMP-1 shedding with the plasma membrane, or LMP-1 secretion in exosomes, may contribute to its observed rapid turnover in these cells. LMP-1 expression must be tightly regulated in EBV-positive lymphoblastoid cells since upwards variation in levels of expression is incompatible with continued cell proliferation (Floettmann et al., 1996
; Hammerschmidt et al., 1989
; Kaykas & Sugden, 2000
). Thus, LMP-1 shedding, or secretion in exosomes, could allow the cell to regulate levels of LMP-1 accumulation. Alternatively, the presence of LMP-1 in extracellular vesicles may serve an immunoregulatory function, as suggested by the observed immunosuppressive effect of purified recombinant LMP-1 protein, or LMP-1-derived peptides, on T cell activation (Dukers et al., 2000
). LMP-1 containing vesicles may deliver LMP-1 or LMP-1 fragments by fusion with infiltrating T cells. An immunosuppressive function is consistent with the cytostatic activity of LMP-1 but is at odds with the observed T cell stimulation induced by EBV-positive lymphoblastoid cell-derived exosomes (Raposo et al., 1996
). Immunoelectron microscopy localization in budding plasma membrane protrusions (Fig. 1C
) argues against the presence of LMP-1 in secreted exosomes, but instead supports the model that LMP-1 is contained in shed plasma membrane microvesicles. However, intracellular membrane structures, such as multivesicular bodies (MVBs), may not have been preserved during the immunolabelling fixation procedure, so the possibility that LMP-1 is secreted in MVB-derived exosomes must also be considered.
LyLMP-1 localization in virus-producing B95-8 cells
Because of its relatively low level of expression at the single-cell level, full-length LMP-1 labelling was undetectable in uninduced B95-8 cells despite the detection of LMP-1 immunoreactivity in B95-8 cells by Western blot analysis (Figs 1A and 3). LyLMP-1, and not LMP-1, is dramatically upregulated upon lytic-cycle induction in B95-8 cells (Baichwal & Sugden, 1987
; Hudson et al., 1985
) and is easily detected by Western blot (Fig. 1A
). Upregulated lyLMP-1 is concentrated in the 1215 % of cells undergoing the lytic cycle (Table 1
), thus facilitating its detection by immunoelectron microscopy at the single-cell level. Restriction of lyLMP-1 labelling to virus-producing cells indicates that lyLMP-1 upregulation upon lytic-cycle induction occurs only in cells in the lytic cycle and does not result from general activation of cell-signalling pathways by TPA/butyrate.
LyLMP-1 labelling was abundant in the nuclear membrane at early stages of the lytic cycle (Fig. 4), and in plasma membranes, cytoplasm and at the site of virus budding from nuclear membranes at later stages of virus production (Fig. 5
). Together, these results suggest a role for lyLMP-1 in virus egress from the cell and/or in lytic cycle progression. The ability of lyLMP-1 to negatively regulate LMP-1 activation of NF-
B (Erickson & Martin, 2000
) further supports a model in which lyLMP-1 contributes to lytic cycle progression, particularly given recent results showing that the activation of NF-
B by LMP-1 plays a role in preventing lytic cycle activation (Adler et al., 2002
; Prince et al., 2002
). Thus, lyLMP-1 may contribute to lytic cycle progression both at a structural level and at the level of regulation of cell signalling.
LyLMP-1 immunoreactivity is associated with extracellular virions in B95-8 virus supernatants (Erickson & Martin, 1997). We have extended these findings by showing the association of lyLMP-1 with intracellular virus particles in the cytoplasm. First attempts to label intracellular and extracellular virus particles with both anti-LMP-1 sera and anti-gp350/220 antibody in thin sections embedded in Lowicryl resin were unsuccessful. However, surface labelling of intracellular virions revealed abundant immunogold labelled lyLMP-1 in intracellular virions (Fig. 7B
). The presence of lyLMP-1 in intracellular virions suggests lyLMP-1 is incorporated into virions as they bud through cellular membranes. Our observations are consistent with previous results suggesting de-envelopment of virus while budding through the outer nuclear membrane followed by re-envelopment upon budding through cytoplasmic or plasma membranes (Gong & Kieff, 1990
). Virus egress from B95-8 cells is likely to involve a process of de-envelopment and re-envelopment in the cytoplasm as the virus particle transits through the endoplasmic reticulum, Golgi and cytoplasmic vesicles (Gong & Kieff, 1990
; Wild et al., 2002
). The process of de- and re-envelopment could culminate in the incorporation of lyLMP-1 into the virion upon final budding through the plasma membrane.
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ACKNOWLEDGEMENTS |
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Received 12 February 2003;
accepted 23 April 2003.