MRC Virology Unit1 and Division of Virology, University of Glasgow2, Institute of Virology, Church Street, Glasgow G11 5JR, UK
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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RSV formation occurs in association with host cell membranes to produce pleomorphic virus particles (Roberts et al., 1995 ). These RSV particles comprise a ribonucleoprotein (RNP) core that is surrounded by a viral envelope in which three different glycoproteins, the attachment (G), fusion (F) and small hydrophobic (SH) proteins are located. The RSV assembly process is still poorly understood and the factors involved in the formation of progeny virus are poorly defined. The involvement of lipid rafts in the assembly of several different members of the Paramyxoviridae has been demonstrated (Ali & Nayak, 2000
; Manie et al., 2000
; Vincent et al., 2000
) and similar membrane platforms may be involved in RSV assembly and maturation.
A particular area of the RSV assembly process that is poorly understood is the role played by host cell proteins. The involvement of F-actin in RSV morphogenesis has been established (Ulloa et al., 1998 ; Burke et al., 1998
) but its precise function in virus assembly has not been defined. In this respect, RSV is not unique since an involvement of the actin network has been demonstrated in several other enveloped viruses where budding occurs at the cell surface (Damsky et al., 1977
; Bohn et al., 1986
; Cudmore et al., 1995
; Sasaki et al., 1995
; Rey et al., 1996
). We have sought to identify other host cell proteins that may be involved in the RSV assembly process. We have identified the integral membrane protein caveolin-1 (cav-1) as a host cell factor that is efficiently incorporated into the mature RSV virion. A number of functions in the host cell have been ascribed to cav-1, which range from providing a scaffolding protein in the formation of caveoli to acting as a negative regulator of several cellular activities (reviewed in Smart et al., 1999
). In this report we have characterized the location of cav-1 within the mature virion and speculate as to the role played by this protein during virus replication.
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Methods |
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Antibodies.
The F protein monoclonal antibody mAb19 was provided by Geraldine Taylor (Institute of Animal Health, Compton, UK). The anti-RSV monoclonal antibody NCL-RSV3 was purchased from Novocastra Laboratories. The caveolin antibody N-20 was purchased from Santa Cruz Laboratories.
Western blotting.
Cell monolayers were either mock-infected or infected with RSV and incubated at 33 °C for 24 h. These monolayers were extracted using 1% SDS, 15% glycerol, 1% -mercaptoethanol, 60 mM sodium phosphate, pH 6·8 and heated at 100 °C for 2 min prior to separation by SDSPAGE and transfer by Western blotting onto a PVDF membrane. After transfer, the membrane was washed with PBSA and blocked for 18 h at 4 °C in PBSA containing 1% Marvel and 0·05% Tween 20. It was then washed twice in PBSA prior to incubation with anti-RSV (1/200 dilution), mAb19 (1/1000 dilution) or anti-cav-1 (1/500 dilution) for 60 min. The membrane was washed four times in PBSA containing 0·05% Tween 20 and probed using either goat anti-mouse or anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma). 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 onto 13 mm glass coverslips and incubated overnight at 37 °C. Cells were mock- or RSV-infected, after which they were fixed with 3% paraformaldehyde in PBS for 30 min at 4 °C. The fixative was removed and the cells washed five times with PBS plus 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 1 h with the secondary antibody, goat anti-mouse or anti-rabbit IgG (whole molecule) conjugated to either FITC or Cy5 conjugate (1/100 dilution) as appropriate. The F-actin network was detected using phalloidinFITC (Sigma). 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.
Scanning electron microscopy (SEM).
Cells were seeded onto 10 mm glass coverslips and incubated overnight at 37 °C. Following infection with RSV for 24 h, the samples were processed as described previously (Sugrue et al., 2001 ) and visualized using a JEOL T100 scanning electron microscope.
Transmission electron microscopy (TEM)
Unicryl embedding.
Cell monolayers were pelleted in BEEM capsules and fixed for 5 h at 4 °C using 0·5% glutaraldehyde in PBS, after which the fixed pellet was washed with PBS. The pellet was dehydrated by transfer through a gradient of 30, 50, 70, 90 and 100% ethanol. The cell pellet was subsequently infiltrated with Unicryl (TAAB Laboratories) and the resin polymerized by UV irradiation at -15 °C. Ultra-thin sections were placed on nickel-coated grids and incubated with either anti-cav-1 (1/100 dilution), mAb19 (1/100 dilution) or anti-RSV (1/50 dilution) for 4 h at 25 °C. The grids were washed with PBS and incubated for 12 h in anti-rabbit IgG (whole molecule) 20 nm colloidal gold (British Biocell International), 10 nm colloidal gold conjugate (Sigma) or anti-mouse IgG (whole molecule) 10 nm colloidal gold conjugate (Sigma) as appropriate. They were then washed in PBS and fixed in osmium tetroxide vapour for 2 h. The samples were then stained using uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.
Epon 812 embedding.
Samples embedded in Epon 812 (TAAB Laboratories) were processed as follows. Cells were pelleted and fixed using 2·5% glutaraldehyde in PBS at 4 °C, post-fixed in 1% osmium tetroxide and washed with PBS. The fixed cells were dehydrated by transfer through a gradient of 30, 50, 70, 90 and 100% ethanol and infiltrated with Epon 812. The resin was polymerized at 70 °C for 48 h and subsequently sectioned. The sections were stained with uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.
Immuno-negative staining of RSV preparations.
RSV-infected cells were detached from a T75 flask together with the tissue culture medium, and harvested. The cell debris was removed by low speed centrifugation (1000 g, 5 min) and the clarified tissue culture medium analysed by TEM. A droplet of the infected tissue culture medium was placed on a Formvar-coated grid and the virus adsorbed for 10 min. Grids were inverted onto a droplet of primary antibody for 4 h, washed in PBS, and then incubated for a further 4 h either with anti-rabbit IgG (whole molecule) 10 nm colloidal gold conjugate (Sigma) or anti-mouse IgG (whole molecule) 5 nm colloidal gold conjugate (Sigma). The grids were washed in PBS and fixed in osmium tetroxide vapour for 2 h. The samples were then stained with uranyl acetate (saturated in 50:50 ethanol/water), washed in PBS and counter-stained with lead citrate.
Samples were examined using a JEOL 1200 EX2 transmission electron microscope.
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Results |
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The surface of RSV-infected cells was examined in detail using SEM (Fig. 1C). Our data clearly show clusters of RSV filaments budding from the cell surface and extending up to 6 µm in length. As in the case of the IF analysis, the filaments appeared to extend across the plane of the apical cell surface. It is likely that many of these filaments that traverse the cell surface are randomly cross-sectioned in the samples prepared for TEM analysis (described below), giving the impression of spherical shaped RSV particles associated with the cell membrane. The surface of RSV-infected Vero cells was additionally examined using in situ sectioning and TEM (data not shown), which confirmed earlier observations that RSV filaments form preferentially at the apical surface of Vero C1008 cells (Roberts et al., 1995
).
We have used TEM to provide a detailed analysis of the RSV maturation process on the cell surface. These studies employed two different embedding techniques. RSV morphology was examined in samples embedded using Epon 812 resin since this allows the best preservation of ultra-structural detail, while Unicryl embedding preserves the antigenicity of the epitopes in the thin sections and was employed for immuno-electron microscopy. Examination of cell sections using immuno-electron microscopy clearly showed the presence of two virus-related structures that were not detected in mock-infected cells and which could be readily identified on the basis of their morphology (Fig. 2A, plate 1). Firstly, there were RSV filaments (VF) protruding from the surface of infected cells and secondly, throughout the cytoplasm we observed the presence of electron-dense IBs, the latter structures being identical to the IBs visualized by confocal microscopy (Fig. 1B
). These structures were of variable size, ranging from approximately 300 nm up to several µm in length. As expected, these structures could not be labelled with either mAb19 or antibodies against the G glycoprotein (data not shown), suggesting the absence of viral glycoprotein. The specific recognition of IBs by anti-RSV (Fig. 2A
, plate 1) suggests that these structures contain RNP-associated proteins. Examination of IBs using TEM at high magnification (Fig. 2A
, plate 2) clearly shows that they are structured rather than being amorphous and contain material whose morphology resembles the RNP structures that are observed by TEM in RSV preparations (see Fig. 5B). The data that we have obtained using TEM show that in the majority of cases the IBs are intimately associated with the RSV filaments that form at the cell surface, and the material which is present within IBs appears to enter the interior of the filaments. However, we have also observed that, in some instances, material from the IBs appears to be associated with the cell membrane in the absence of RSV filaments (Fig. 2A
, plate 1, highlighted by open box). It is interesting to note that structures resembling IBs could be detected by confocal microscopy on the surface of some non-permeabilized cells using anti-RSV (data not shown). The significance of the latter observation is at present unclear but suggests that the RNP cores may make direct contact with the cell exterior. It is likely that the dimensions of the RSV filaments (approximately 0·15x6 µm) may make them fragile and prone to detachment from the cell surface, consequently leaving the remaining underlying IB-associated material on the cell surface. Alternatively, RSV filaments may be detached from the cell surface as a consequence of virus release following maturation. However, we have no evidence that free RNP cores were present in the tissue culture medium of RSV-infected cells.
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2. The cell distribution of cav-1 is altered by RSV infection
A relatively unexplored aspect of RSV assembly concerns the role of host cell factors in the assembly process. Since the filaments are clearly visible by light microscopy (Parry et al., 1979 ), IF microscopy affords a relatively simple assay to determine which, if any, host cell proteins associate with mature RSV. In our assay, the host cell protein cav-1 showed a significant change in fluorescence staining upon RSV infection. In mock-infected cells, the observed staining pattern for cav-1 consisted of small red spots (Fig. 3A
, plate 1). However, in RSV-infected cells, the cav-1 adopted a staining pattern that was composed of the small spots, similar to that observed in mock-infected cells, and a filamentous staining pattern (Fig. 3A
, plates 2 and 3) that was not seen in mock-infected cells. The filamentous staining pattern appeared to resemble the RSV filaments that were visualized in infected cells using mAb19 and anti-RSV (Fig. 1B
). This filamentous form of cav-1 appeared to align with the direction of the green-stained actin filaments (F-actin) when the latter were visualized following phalloidinFITC staining of F-actin. We also noted that the number of visible actin filaments appeared to increase in the RSV-infected cells when compared with those observed in mock-infected cells, which agrees with recent observations (Gower et al., 2001
). However, the filamentous cav-1 that we observed in RSV-infected cells showed no significant co-localization with F-actin. Analysis of mock- and RSV-infected monolayers by Western blotting using anti-cav-1 (Fig. 3A
, plate 4) showed the presence of a single 23 kDa protein band, which is the expected size for cav-1. In addition Western blotting showed that the detectable levels of cav-1 remained unchanged following introduction of the virus, suggesting that RSV does not stimulate cav-1 expression, but rather, the cell surface distribution of cav-1 is altered during infection.
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The association of cav-1 with RSV filaments was further examined by immuno-electron microscopy (Fig. 5). In this study, two strategies were employed to prepare samples for analysis, namely thin sections obtained from RSV-infected cells (Fig. 5A
) and immuno-negative staining of RSV particles that were present in the tissue culture medium (Fig. 5B
). Examination of RSV-infected cell sections showed the labelling of RSV with anti-cav-1 (Fig. 5A
, plates 1 and 2), as evidenced by the presence of the 10 nm gold probe on the morphologically distinct RSV filaments. In addition, we observed that the pattern of labelling was localized in specific areas of the viral filaments, visualized as between six and 12 tightly clustered 10 nm gold particles. We also observed a clustering of gold particles in specific regions of the infected cell plasma membrane, which may represent regions of cav-1 enrichment such as those present within the caveoli or lipid rafts. In this respect it is interesting to note that clusters of gold particles (i.e. cav-1 enrichment) generally appeared in regions of the plasma membrane that were close to RSV filament formation. Further analysis of mature cell-associated RSV filaments by double immuno-gold labelling showed co-localization of anti-RSV and anti-cav-1 (Fig. 5A
, plate 3). Additionally, cav-1 appeared to be associated with the RSV filaments early in the assembly process, since early budding structures were labelled with anti-cav-1 (Fig. 5A
, plate 4). This localized labelling of RSV filaments by anti-cav-1 using TEM is consistent with the observed localized staining pattern of cav-1 on RSV filaments by confocal microscopy.
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Discussion |
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Caveoli have been shown to provide a mechanism by which RSV can enter the host cell following cell attachment (Werling et al., 1999 ). However, our data suggest that the major component of caveoli structures, namely cav-1, is additionally involved in the subsequent steps of virus assembly. The results of immuno-electron microscopy showed that RSV filaments generally form at, or in close proximity to, regions on the cell surface that are strongly labelled by anti-cav-1. Furthermore, we observed that the maturing RSV could be labelled with anti-cav-1 soon after the budding process was initiated. This suggests that cav-1 may be an important host cell factor which is involved either in the targeting of RSV envelope proteins to the cell surface or as a focus point for the initiation of RSV assembly on the cell surface membrane. In addition, our results clearly show that cav-1 is subsequently incorporated into the envelope of maturing RSV in clusters along the length of the RSV filaments. The route by which cav-1 is incorporated into maturing RSV particles remains to be established. It is not clear if cav-1 incorporation occurs passively during virus maturation or actively by association with either host cell- or virus-specific proteins. However, the incorporation of host cell proteins into mature virus is not unique to RSV, since this phenomenon has been documented both in vesicular stomatitis virus (Lodish & Porter, 1980
; Calafat et al., 1983
) and human immunodeficiency virus 1 (Saifuddin et al., 1994
; Marschang et al., 1995
). In addition, a recent study has shown that several intracellular signalling molecules can be incorporated into virus particles (Pickl et al., 2001
). These previous reports provide a precedent for our observation that cav-1 can be incorporated into the envelope of mature RSV.
We have noted that in RSV-infected cells, the cav-1 filaments appear to align with F-actin, although no direct interaction was identified, but it is possible that such an interaction may involve other actin-associated proteins. Recent studies have shown that Rho activation leads to the association of cav-1 with actin stress fibres via the actin-associated protein, filamin (Stahlhut & van Deurs, 2000 ). It is not clear if a similar mechanism operates during RSV assembly since we have so far failed to identify any stable interactions between filamin and either RSV filaments or cav-1 in RSV-infected cells (data not shown). However, the actin network may provide a mechanism, either directly or indirectly, whereby specific virus and host cell factors are recruited into maturing RSV.
The nature of the interaction between cav-1 and the mature RSV filaments is currently not defined. It has been established that cav-1 is an integral membrane protein (Kurzchalia et al., 1994 ) and as such has the potential to be inserted directly into the RSV envelope. In addition, we have failed to detect any stable interaction between cav-1 and either the F or G glycoproteins (data not shown). However, if such an interaction exists, it may be mediated indirectly by one or more host cell factor. Previous reports have shown that cav-1 is able to interact with RhoA GTPase (Gingras et al., 1998
), a molecule which is activated during RSV infection (Gower et al., 2001
) and which is able to interact with the F1 subunit of the F glycoprotein (Pastey et al., 1999
).
A final question concerns the biological consequences of cav-1 incorporation into mature RSV particles. The role that RSV-associated cav-1 plays in the virus replication cycle and the effect of its presence on RSV pathogenesis is currently undefined. Although cav-1 is the major structural component of caveoli, several other specific functions have been ascribed for cav-1, such as regulating signalling pathways in the cell. Evidence has suggested that cav-1 can down-regulate steps in the p42/44 MAPK signal transduction pathway (Engelman et al., 1998 ; Galbiati et al., 1998
) and the negative regulation of nitric oxide synthase by cav-1 has been demonstrated (Garcia-Cardena et al., 1997
; Ju et al., 1997
; Ghosh et al., 1998
). It may be speculated that down-regulating specific host cell activities and signalling pathways could be advantageous to the virus either during the initial stages of virus entry or during the assembly process.
Taken as a whole, the data presented in this report suggest that the RSV assembly process is complicated, involving virus structural proteins as well as at least one specific host cell membrane-associated factor. Additionally, this is the first report of a cellular protein that is incorporated into the RSV envelope during the assembly process. Although we have shown that cav-1 is a host cell-derived factor that is incorporated into mature RSV, its role during virus infection remains to be established. In addition, it is not clear if cav-1 is a component of other paramyxoviruses, particularly those that mature as filaments on the infected cell surface.
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Acknowledgments |
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References |
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Received 29 August 2001;
accepted 16 November 2001.