MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK1
Author for correspondence: Richard Sugrue. Fax +44 141 337 2236. e-mail r.sugrue{at}vir.gla.ac.uk
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Abstract |
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Introduction |
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The mammalian caveolin gene family consists of three members which are referred to as caveolin-1, -2 and -3 (Smart et al., 1999 ). Whereas caveolins-1 and -2 are expressed in several cell lines (e.g. endothelial and fibroblastic), caveolin-3 expression is restricted to striated muscle cells. We have previously reported that caveolin-1 (cav-1) associates with the immature RSV particle at the plasma membrane and that this protein is subsequently incorporated into the mature virion (Brown et al., 2002
). It has been established by several groups that cav-1 is intimately associated with specialized lipid-raft structures on the host-cell membrane, including caveolae. In this report we have further characterized the sites of RSV assembly.
Since RSV interacts with cav-1, we were interested to determine if the raft-specific ganglioside GM1 was similarly involved in virus assembly. In addition, we investigated the effect of virus replication on the tyrosine phosphorylated form of caveolin-1 (pcav-1). Cav-1 was first identified as a major tyrosine phosphorylated protein in v-Src-transformed cells (Glenney & Zokas, 1989 ). Recent work by Lee et al. (2000)
has shown that tyrosine phosphorylation enables binding of growth hormone receptor binding protein 7 (Grb7), an Src homology (SH) 2 domain-containing adapter protein, to cav-1. Such SH 2 adapter proteins are able to link a variety of catalytic subunits to tyrosine-specific phosphorylated receptors or intermediates in signal transduction pathways. Several cellular processes are thought to be regulated by pcav-1 (Smart et al., 1999
) and the formation of the pcav-1/Grb7 signalling complex has been implicated in cell transformation. Given the role that pcav-1 plays in modulating the cell's physiology we were also interested to determine if, and to what extent, RSV replication altered the pattern of pcav-1 expression.
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Methods |
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Antibodies.
The F and G protein monoclonal antibodies, mAb19 and mAb30 respectively, were provided by Geraldine Taylor (IAH, Compton, UK. The anti-Grb7 (sc 607) was purchased from Santa Cruz Laboratories. The anti-caveolin-1 (cat. no. 610059) and phosphocaveolin (PY14) antibodies were purchased from BD Transduction Laboratories.
Western blotting.
Mock- and RSV-infected cell monolayers were extracted with boiling loading buffer (1% SDS, 15% glycerol, 1% -mercaptoethanol, 60 mM sodium phosphate, 1 mM sodium orthovanadate, 5 mM sodium fluoride, pH 6·8) and then heated at 100 °C for 5 min. The lysates were separated by SDSPAGE and transferred by Western blotting onto a PVDF membrane. After transfer, the membrane was washed with PBSA and blocked for 1 h at 20 °C in PBSA containing 1% BSA and 0·05% Tween 20 prior to probing with PY14 (1/500 dilution) for 18 h at 4 °C. The membrane was subsequently processed as previously described (Brown et al., 2002
) and the protein bands were visualized using the ECL protein detection system (Amersham). Apparent molecular masses were estimated using Rainbow protein markers (Amersham) in the molecular mass range 14·3220 kDa.
Immunofluorescence.
Cells were seeded on 13 mm glass coverslips and incubated overnight at 37 °C. Cells were mock- or RSV-infected and incubated at 33 °C for 18 h, unless otherwise stated, and fixed with 3% paraformaldehyde in PBS for 30 min at 4 °C. The fixative was removed and the cells were washed five times with PBS+1 mM glycine and once with PBS. The cells were either examined directly or permeabilized using 0·1% saponin in PBS. Following incubation at 25 °C for 1 h with the primary antibody, the cells were washed and incubated for a further hour with the secondary antibody, goat anti-mouse or anti-rabbit IgG (whole molecule) conjugated to either FITC or Cy5 (1/100 dilution) as appropriate. The F-actin network was detected using phalloidinFITC (Sigma) as described previously (Brown et al., 2002 ). The GM1 microdomains were visualized using cholera toxin B subunit conjugated to FITC (CTX-BFITC). The CTX-BFITC (Sigma) was prepared as a stock solution (1 mg/ml) in distilled sterilized water. RSV-infected cells were incubated with 4 µg/ml CTX-BFITC for 20 min at 4 °C after which they were washed using PBSA and fixed with paraformaldehyde. The CTX-BFITC-labelled cells were then probed with specific antibodies as described above.
In each case, the stained cells were mounted on slides using Citifluor and visualized using a Zeiss Axioplan 2 confocal microscope. The images were processed using LSM 510 v2.01 software.
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Results |
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Analysis of mock-infected cells by confocal microscopy showed a punctate pattern of staining, while in RSV-infected cells the mature RSV filaments were efficiently labelled with anti-cav-1, as described previously (Brown et al., 2002 ), thus confirming the presence of RSV-associated cav-1 (Fig. 1A
). This was evidenced by the efficient colocalization (yellow) of mAb19 (green) and anti-cav-1 (red) in the readily visible surface filaments. As we have noted previously, the labelling of mature RSV with mAb19 was confined to the virus structures, whereas labelling with anti-cav-1 was observed on both the cell membrane and virus filaments.
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Altered distribution of pcav-1 in RSV-infected Vero cells
We further examined the effect of RSV infection on the cellular distribution of two alternative forms of caveolin that are expressed in epithelial cells, namely caveolin-2 (cav-2) and tyrosine phosphorylated cav-1. Studies have shown that cav-1 is able to interact with cav-2 to form a hetero-oligomeric protein complex that is subsequently transported from the Golgi complex to specific compartments at the cell surface (Scheiffele et al., 1998 ). This suggested to us that cav-2 may be incorporated into mature RSV filaments in a manner similar to that observed for cav-1. However, we observed no significant change in the cellular distribution of cav-2 in response to RSV-infection (data not shown), indicating the absence of its association with the mature virus.
Several studies have shown that cav-1 can be modified by tyrosine phosphorylation at tyrosine 14. This form of cav-1 has been implicated in several signalling events (reviewed in Smart et al., 1999 ) and its presence can be specifically detected using the monoclonal antibody PY14. The use of this reagent has been described in several publications which have investigated the biochemical properties of phosphocaveolin-1 (Lee et al., 2000
; Caselli et al., 2001
; Volonte et al., 2001
). In this report, the tyrosine phosphorylated form of cav-1 will be referred to as pcav-1.
We observed a significant change in the distribution of pcav-1 during RSV-infection (Fig. 2A). In mock-infected cells, PY14 labelling resulted in a staining pattern that was essentially confined to the cell surface or its immediate vicinity. In contrast, we noted that in RSV-infected cells, while the pattern of surface staining remained similar to that in mock-infected cells, the intensity of surface labelling was significantly less. Analysis of the internal distribution of pcav-1 using PY14 showed the appearance of clusters of relatively long cytoplasmic vesicles in virus-infected cells, which were not observed in mock-infected cells. Although these structures appeared to be significantly brighter that the surface-labelled pcav-1 (observed in mock-infected cells), analysis of pcav-1 levels by Western blotting suggested no significant increase in pcav-1 levels in RSV-infected cells (Fig. 2B
). In this assay, pcav-1 appeared as a closely spaced doublet band of 25 and 28 kDa. This has been noted previously and may arise from variations in the degree to which pcav-1 is modified by serine phosphorylation (Nomura & Fujimoto, 1999
; Schlegel et al., 2001
). However, the significant reduction in the surface-labelled pcav-1 suggests that these structures result from a redistribution of pcav-1 from the cell surface into the interior of the cell following RSV infection, rather than an increase in the pcav-1 levels.
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Analysis of the cytoplasmic levels of pcav-1 during the course of infection was examined (Fig. 4). This showed that the change in surface staining was not immediate following RSV binding and entry and did not occur early in the infection, but became clearly visible between 9 and 18 h post-infection. This coincides with the approximate time of RSV filament formation that has been previously observed in Vero C1008 cells (Roberts et al., 1995
; Brown et al., 2002
), suggesting that in infected cells, there is a decrease in pcav-1 levels associated with the FA structures which appears to correlate with cytoplasmic vesicle formation. The data also suggest that this pcav-1 redistribution may be related to the formation of RSV filaments.
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Discussion |
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Our results suggest that, in addition to cav-1, the glycosphingolipid GM1 may also be subsequently incorporated into the mature virion during assembly. Lipid-raft membrane compartments within the plasma membrane form highly ordered structures, compared to the bulk of the phospholipid in the cell membrane which is thought to exist in a liquid crystalline phase (Brown & London, 1998 ). The lipid composition of a membrane therefore has important structural and functional consequences. Our results suggest that such ordered microdomains, which are enriched in GM1, may be present within the viral envelope of the mature RSV. The incorporation of cholesterol-dependent raft structures into the Influenza virus envelope correlates with a reduction in the fluidity' of the viral envelope (Scheiffele et al., 1999
) thus demonstrating the influence of lipid composition on the properties of the envelope. The incorporation of GM1 into the RSV membrane may therefore impart biophysical properties to the viral membrane that may have important consequences for the stability of the lipid envelope that surrounds the mature virion. However, the precise functional significance of the RSV-associated raft components within the mature virus is presently unclear, but their presence may have implications for the development and the design of novel antiviral strategies.
In addition, our data clearly suggest that RSV assembly coincides with a cellular redistribution of pcav-1 from locations that are either near to, or within, focal adhesions, into large cytoplasmic vesicles. In our analysis we noted relatively high levels of pcav-1 in mock-infected polarized Vero cells, compared to those in several other established cell lines (e.g. BHK). The level of detectable pcav-1 in each specific cell line is likely to be dependent upon the relative kinetics of cav-1 phosphorylation and dephosphorylation, processes which are mediated by specific cellular kinases and phosphatases. Tyrosine phosphorylation of cav-1 was first demonstrated in transformed cell lines, but the presence of pcav-1 has been demonstrated in normal, untransformed cells (Aoki et al., 1999 ). However, although the clinical significance of the redistribution of pcav-1 in RSV-infected cells is presently unclear, tyrosine phosphorylation of caveolin-1 has been shown to be involved in several different cellular processes such as osmotic sensoring (Volonte et al., 2001
) and anchorage-independent growth (Engelman et al., 1997
). Although these are very distinct biochemical processes, they are both characterized by structural changes in the cellular cytoskeleton. Recent evidence suggests that pcav-1 is involved in the generation of aggregated caveolae (Nomura & Fujimoto, 1999
) which manifest themselves in the form of large cytoplasmic vesicles. This process is mediated by the rearrangement of the cellular cytoskeleton (Fujimoto et al., 1995
). A role for actin in the morphogenesis of several viruses has been reported (Damsky et al., 1977
; Bohn et al., 1986
; Cudmore et al., 1995
; Sasaki et al., 1995
; Rey et al., 1996
) and a similar role for actin in RSV morphogenesis has been suggested. The latter is based primarily on the observation that RSV assembly is inhibited by reagents that interfere with actin polymerization (Ulloa et al., 1998
; Burke et al., 1998
). In this regard, the formation of actin stress fibres has been reported during RSV assembly (Gower et al., 2001
) and presumably reflects some changes in the structure of the actin cytoskeleton. However, the precise molecular mechanisms by which actin is involved in the assembly process have yet to be established.
Our data provide further evidence that RSV assembly is accompanied by some form of structural rearrangement within the cytoskeletal network, which coincides both with a cellular redistribution of pcav-1 and the formation of cytoplasmic vesicles containing both pcav-1 and Grb7. Although the downstream effects of Grb7 on specific cellular processes within the cell are currently poorly defined (reviewed in Han et al., 2001 ), the formation of a pcav-1/Grb7 signalling complex has been shown to augment anchorage-independent growth in tissue culture (Lee et al., 2000
). In this regard, it is interesting to note that anchorage-independent growth is associated with a change in the cytoskeletal structure of the cell (Pawlak & Helfman, 2001
). It is therefore possible that an interaction between pcav-1 and Grb7 may be part of the biochemical mechanism whereby RSV can utilize the host-cell's cytoskeletal network during virus assembly.
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Acknowledgments |
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References |
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Received 7 February 2002;
accepted 28 February 2002.